Senscent cell-derived inhibitors of DNA synthesis

ABSTRACT

The use of liposomal formulations, particularly formulations of positively charged and neutral lipids facilitates cellular uptake of SDI molecules. The transcription and/or expression of SDI-1-encoding nucleic acid molecules is facilitated by constructs that contain intervening untranslated regions.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of PCT US94/09700 (filed Aug.26, 1994), which is a continuation-in-part of U.S. patent applicationSer. No. 08/274,535 (filed Jul. 13, 1994) now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 08/229,420(filed Apr. 15, 1994), now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 08/203,535 (filed Feb. 25, 1994), nowabandoned, which is a continuation-in-part of U.S. patent applicationSer. No. 08/153,564 (filed Nov. 17, 1993), now abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 08/113,372(filed Aug. 30, 1993), now abandoned, which is a continuation-in-part ofU.S. patent application Ser. No. 07/970,462 (filed Nov. 2, 1992, andissued as U.S. Pat. No. 5,302,706 on Apr. 12, 1994); and divisional U.S.patent application Ser. No. 08/160,814 (filed Jan. 3, 1994,), now U.S.Pat. No. 5,424,400, and Ser. No. 08/268,439 (filed Jun. 30, 1994) nowabandoned, all of which Applications are continuations-in-part of U.S.patent application Ser. No. 07/808,523 (filed Dec. 16, 1991, nowabandoned).

FIELD OF THE INVENTION

The present invention is in the field of recombinant DNA technology.This invention is directed to a gene sequence and a protein that effectsthe ability of cells to become senescent. This invention was supportedwith Government funds. The Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Normal human diploid cells have a finite potential for proliferativegrowth (Hayflick, L. et al., Exp. Cell Res. 25:585 (1961); Hayflick, L.,Exp. Cell Res. 37:614 (1965)). Indeed, under controlled conditions invitro cultured human cells can maximally proliferate only to about 80cumulative population doublings. The proliferative potential of suchcells has been found to be a function of the number of cumulativepopulation doublings which the cell has undergone (Hayflick, L. et al.,Exp. Cell Res. 25: 585 (1961); Hayflick, L. et al., Exp. Cell Res. 37:614 (1985)). This potential is also inversely proportional to the invivo age of the cell donor (Martin, G. M. et al., Lab. Invest. 23:86(1979); Goldstein, S. et al., Proc. Natl. Acad. Sci. (U.S.A.) 64:155(1969); Schneider, E. L., Proc. Natl. Acad. Sci. (U.S.A.) 73:3584(1976); LeGuilty, Y. et al., Gereontologia 19:303 (1973)).

Cells that have exhausted their potential for proliferative growth aresaid to have undergone “senescence.” Cellular senescence in vitro isexhibited by morphological changes and is accompanied by the failure ofa cell to respond to exogenous growth factors. Cellular senescence,thus, represents a loss of the proliferative potential of the cell.Although a variety of theories have been proposed to explain thephenomenon of cellular senescence in vitro, experimental evidencesuggests that the age-dependent loss of proliferative potential may bethe function of a genetic program (Orgel, L. E., Proc. Natl. Acad. Sci.(U.S.A.) 49:517 (1963); De Mars, R. et al., Human Genet. 16:87 (1972);M. Buchwald, Mutat. Res. 44:401 (1977); Martin, G. M. et al., Amer. J.Pathol. 74:137 (1974); Smith, J. R. et al., Mech. Age. Dev. 13:387(1980); Kirkwood, T. B. L. et al., Theor. Biol. 53:481 (1975).

Cell fusion studies with human fibroblasts in vitro have demonstratedthat the quiescent phenotype of cellular senescence is dominant over theproliferative phenotype (Pereira-Smith, O. M et al., Somat. Cell Genet.8:731 (1982); Norwood, T. H. et al., Proc. Natl. Acad. Sci. (U.S.A.)71:223 (1974); Stein, G. H. et al., Exp. Cell Res. 130:155 (1979)).

Insight into the phenomenon of senescence has been gained from studiesin which senescent and young (i.e. non-senescent) cells have been fusedto form heterodikaryons. In order to induce senescence in the “young”nucleus of the heterodikaryon (as determined by an inhibition in thesynthesis of DNA), protein synthesis must occur in the senescent cellprior to fusion (Burmer, G. C. et al., J. Cell. Biol. 94:187 (1982);Drescher-Lincoln, C. K. et al., Exp. Cell Res. 144:455 (1983); Burner,G. C. et al., Exp. Cell Res. 145:708 (1983); Drescher-Lincoln, C. K. etal., Exp. Cell Res. 153:208 (1984).

Likewise, microinjection of senescent fibroblast mRNA into youngfibroblasts has been found to inhibit the ability of the young cells tosynthesize DNA (Lumpkin, C. K. et al., Science 232:393 (1986)).Researchers have identified unique mRNAs that are amplified in senescentcells in vitro (West, M. D. et al., Exp. Cell Res. 184:138 (1989);Giordano, T. et al., Exp. Cell Res. 185:399 (1989)).

The human diploid endothelial cell presents an alternative cell type forthe study of cellular senescence because such cells mimic cellularsenescence in vitro (Maciag, T. et al., J. Cell. Biol. 91:420 (1981);Gordon, P. B. et al., In Vitro 19:661 (1983); Johnson, A. et al., MechAge. Dev. 18:1 (1982); Thornton, S. C. et al., Science 222:623 (1983);Van Hinsbergh, V. W. M. et al., Eur. J. Cell Biol. 42:101 (1986);Nichols, W. W. et al., J. Cell. Physiol. 132:453 (1987)).

In addition, the human endothelial cell is capable of expressing avariety of functional and reversible phenotypes. The endothelial cellexhibits several quiescent and non-terminal differentiation phenotypes(Folkman, J. et al., Nature 288:551 (1980); Maciag, T. et al., J. CellBiol. 94:511 (1982); Madri. J. A. et al., J. Cell Biol. 97:153 (1983);Montesano, R., J. Cell Biol. 99:1706 (1984); Montesano, R. et al., J.Cell Physiol. 34:460 (1988)).

It has been suggested that the pathway of human cell differentiation invitro involves the induction of cellular quiescence mediated bycytokines that inhibit growth factor-induced endothelial cellproliferation in vitro (Jay, M. et al., Science 228:882 (1985); Madri,J. A. et al., In Vitro 23:387 (1987); Kubota, Y. et al., J. Cell Biol.107:1589 (1988); Ingber, D. E. et al., J. Cell Biol. 107:317 (1989)).

Inhibitors of endothelial cell proliferation also function as regulatorsof immediate-early transcriptional events induced during the endothelialcell differentiation in vitro, which involves formation of thecapillary-like, tubular endothelial cell phenotype (Maciag, T., In: Imp.Adv. Oncol. (De Vita, V. T. et al., eds., J. B. Lippincott.Philadelphia, 42 (1990); Goldgaber, D. et al., Proc. Natl. Acad. Sci.(U.S.A.) 86:7606 (1990); Hla, T. et al., Biochem. Biophys. Res. Commun.167:637 (1990)). The inhibitors of cell proliferation include:

1. Interleukin-1a (IL-1a) (Montesano, R. et al., J. Cell Biol. 99:1706(1984); Montesano, R. et al., J. Cell Physiol. 122:424 (1985); Maciag,T. et al. (Science 249:1570-1574 (1990));

2. Tumor necrosis factor (Frater-Schroder, M. et al., Proc. Natl. Acad.Sci. (U.S.A.) 84:5277 (1987); Sato, N. et al., J. Natl. Cancer Inst.76:1113 (1986); Pber, J. P., Amer. J. Pathol. 133:426 (1988); Shimada,Y. et al., J. Cell Physiol. 142:31 (1990));

3. Transforming growth factor-β (Baird, A. et al., Biochem. Biophys.Res. Commun. 138:476 (1986); Mullew, G. et al., Proc. Natl. Acad. Sci.(U.S.A.) 84:5600 (1987); Mairi, J. A. et al., J. Cell Biol. 106:1375(1988));

4. Gamma-interferon (Friesel, R. et al., J. Cell Biol. 104:689 (1987);Tsuruoka, N. et al., Biochem. Biophys. Res. Commun. 155:429 (1988)) and

5. The tumor promoter, phorbol myristic acid (PMA) (Montesano, R. etal., Cell 42:469 (1985); Doctrow, S. R. et al., J. Cell Biol. 104:679(1987); Montesano, R. et al., J. Cell. Physiol. 30 130:284 (1987);Hoshi, H. et al., FASAB J. 2:2797 (1988)).

The prospect of reversing senescence and restoring the proliferativepotential of cells has implications in many fields of endeavor. Many ofthe diseases of old age are associated with the loss of this potential.Restoration of this ability would have far-reaching implications for thetreatment of this disease, of other age-related disorders, and, of agingper se.

In addition, the restoration of proliferative potential of culturedcells has uses in medicine and in the pharmaceutical industry. Theability to immortalize nontransformed cells can be used to generate anendless supply of certain tissues and also of cellular products.

The significance of cellular senescence has accordingly been appreciatedfor several years (Smith, J. R., Cellular Ageing, In: Monographs inDevelopmental Biology, Sauer, H. W. (Ed.), S. Karger, New York, N.Y.17:193-208 (1984); Smith, J. R. et al. Exper. Gerontol. 24:377-381(1989), herein incorporated by reference). Researchers have attempted toclone genes relevant to cellular senescence. A correlation between theexistence of an inhibitor of DNA synthesis and the phenomenon ofcellular senescence has been recognized (Spiering, A. I. et al, Exper.Cell Res. 179:159-167 (1988); Pereira-Smith, O. M. et al., Exper. CellRes. 160:297-306 (1985); Drescher-Lincoln, C. K. et al., Exper. CellRes. 153:208-217 (1984); Drescher-Lincoln, C. K. et al., Exper. CellRes. 144:455-462 (1983)). Moreover, the relative abundance of certainsenescence-associated RNA molecules has been identified (Lumpkin, C. K.et al., Science 232:393-395 (1986)).

Several laboratories have used the “subtraction-differential” screeningmethod to identify cDNA molecules derived from RNA species that arepreferentially present in senescent cells (Kleinsek, D. A., Age 12:55-60(1989); Giordano, T. et al., Exper. Cell. Res. 185:399-406 (1989);Sierra, F. et al., Molec. Cell. Biol. 9:5610-5616 (1989); Pereira-Smith,O. M. et al., J Cell. Biochem. (Suppl 0 (12 part A)) 193 (1988);Kleinsek, D. A., Smith, J. R., Age 10:125 (1987)).

In one method, termed “subtraction-differential” screening, a pool ofcDNA molecules is created from senescent cells, and then hybridized tocDNA or RNA of growing cells in order to “subtract out” those cDNAmolecules that are complementary to nucleic acid molecules present ingrowing cells. Although useful, for certain purposes, the“subtraction-differential” method suffers from the fact that it is notpossible to determine whether a senescence-associated cDNA molecule isassociated with the cause of senescence, or is produced as a result ofsenescence. Indeed, many of the sequences identified in this manner havebeen found to encode proteins of the extra-cellular matrix. Changes inthe expression of such proteins would be unlikely to cause senescence.

SUMMARY OF THE INVENTION

This application is a continuation-in-part of PCT US94/09700 (filed Aug.26, 1994), herein incorporated by reference in its entirety. The presentinvention concerns, in part, the observation that normal human cellsexhibit a limited replicative potential in vitro and become senescentafter a certain number of divisions. As the cells become senescent, theyshow several morphological and biochemical changes, such as enlargementof cell size, changes of extracellular matrix components,unresponsiveness to mitogen stimulation and failure to express growthregulated genes.

The present invention identifies an inhibitor of DNA synthesis that isproduced in senescent cells. This inhibitor plays a crucial role in theexpression of the senescent phenotype. The gene coding for the inhibitorwas identified by incorporating a senescent cell cDNA library into amammalian expression vector. The cDNA library was then transfected intoyoung, cycling cells to identify those library members that suppressedthe initiation of DNA synthesis.

Efficient DEAE dextran-mediated transfection enabled the isolation ofputative senescent cell derived inhibitor (SDI) sequences in threedistinct cDNA clones. The expression of one (SDI-1) increased 20 fold atcellular senescence, whereas that of the others (SDI-2 and SDI-3)remained constant.

In summary, the present invention achieves the cloning of an inhibitorof DNA synthesis using a functional assay. This method may be applied toclone other genes involved in negative regulation of the cell cycle,such as tissue specific differentiation and tumor suppression genes.Using this method, three inhibitor sequences have been cloned. One ofthese sequences (SDI-1) appears to be closely related to cellularsenescence.

In detail, the invention provides a nucleic acid molecule that encodes aprotein or polypeptide capable of inhibiting DNA synthesis in arecipient cell.

In particular, the invention provides a liposome preparation thatcomprises an SDI molecule, and particularly one that comprises:

(a) a mixture of a polycationic and a neutral lipid; and

(b) an SDI molecule selected from the group consisting of SDI-1 proteinand an SDI-1-encoding nucleic acid molecule.

The invention particularly provides such liposome preparations whereinthe polycationic lipid is2,3-dioleyloxy-N-[2(sperminecarboxamido)-ethyl]-N,N-dimethyl-1-propanaminium-trifluoroacetate(DOSPA) and/or wherein the neutral lipid isdioleolyphosphatidylethanolamine (DOPE).

The invention particularly concerns the embodiments wherein (A) the SDImolecule is an SDI-1-encoding nucleic acid molecule that is operablylinked to a promoter, but is separated from the operably linked promoterby a non-translated intervening polynucleotide and/or (B) wherein theSDI-1-encoding nucleic acid molecule is operably linked to a promoter,and contains a non-translated intervening polynucleotide which separatesa region of the SDI-1-encoding nucleic acid that encodes part of SDI-1from a region of the SDI-1-encoding nucleic acid that encodes adifferent part of SDI-1.

The invention also provides a method for preparing a liposomepreparation of SDI molecules which comprises incubating liposomes thatcomprise a mixture of a polycationic and a neutral lipid with an SDImolecule selected from the group consisting of SDI-1 protein and anSDI-1-encoding nucleic acid molecule.

The invention also concerns a method of providing an SDI molecule to acell which comprises:

(A) contacting the cell with a liposome preparation that comprises amixture of a polycationic and a neutral lipid and an SDI moleculeselected from the group consisting of SDI-1 protein and anSDI-1-encoding nucleic acid molecule; and

(B) permitting the intracellular delivery of the SDI-1 molecule of theliposome preparation.

The invention also concerns an SDI-1-encoding nucleic acid molecule,operably linked to a promoter, but separated from the operably linkedpromoter by a non-translated intervening polynucleotide.

The invention also provides a method of transcribing an SDI-1-encodingnucleic acid molecule which comprises:

(A) providing to a cell the SDI-1-encoding nucleic acid molecule,operably linked to a promoter, but separated from the operably linkedpromoter by a non-translated intervening polynucleotide; and

(B) permitting the promoter to mediate the transcription of theSDI-1-encoding nucleic acid molecule.

The invention also provides a nucleic acid molecule that encodes SDI-1,a fragment of SDI-1, an SDI-1 fusion protein or a mimetic or analog ofSDI-1.

The invention also provides a protein or polypeptide capable ofinhibiting DNA synthesis in a recipient cell, wherein the protein orpolypeptide is SDI-1, a fragment of SDI-1, an SDI-1 fusion protein or amimetic or analog of SDI-1.

The invention additionally provides a method for treating a disease ofundesired cellular proliferation which comprises providing to arecipient a nucleic acid molecule that encodes SDI-1, a fragment ofSDI-1, an SDI-1 fusion protein, an SDI-1 mimetic, or an analog of SDI-1.

The invention additionally provides a method for treating a disease ofundesired cellular proliferation which comprises providing to arecipient a protein or polypeptide capable of inhibiting DNA synthesisin a recipient cell, wherein the protein or polypeptide is SDI-1, afragment of SDI-1, an SDI-1 fusion protein or a mimetic or analog ofSDI-1.

The invention additionally provides a method for treating a disease ofundesired cellular quiescence which comprises providing to a recipient anucleic acid molecule that encodes an inhibitor of SDI-1.

The invention additionally provides a method for treating a disease ofundesired cellular quiescence which comprises providing to a recipient aprotein, polypeptide, or organic molecule capable of inhibiting SDI-1.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the structure of the cDNA cloning and expression vector,pcDSRαΔ (B represents BamHI site).

FIGS. 2A, 2B and 2C identify cDNA clones inhibitory to young cell DNAsynthesis. The different bars represent independent transfectionexperiments for individual plasmids, * indicates not done, a negativenumber indicates labeling indices higher than the controls. Each graphshows results from a different pool of plasmids.

FIG. 3 shows antisense SDI cDNA transfection. Antisense cDNA expressionplasmids were made and co-transfected with pCMVβ into young cells. Lane1: control pcDSRαΔ, lane 2: pcDSRαΔ-SDI-1, lane 3: pcDSRαΔ antiSDI-1,lane 4: pcDSRαΔ-SDI-2, lane 5: pcDSRαΔ- antiSDI-2.

FIG. 4 shows the changes in poly A+RNA recovery from total RNA duringcellular aging.

FIGS. 5A-5D provide the nucleotide (SEQ. ID NO: 1) and amino acid (SEQID NO: 2) sequences of SDI-1 cDNA.

FIG. 6 shows the kinetics of SDI-1 induction in young cells (populationdoubling 28) obtained following exposure to either 4 Gy of γ-irradiation(open circles) or 400 μM hydrogen peroxide for 1 hour (closed circles).

DETAILED DESCRIPTION OF THE INVENTION

I. Cellular Senescence

Replicative senescence of normal human diploid fibroblasts in culture isa well established and widely accepted model for cellular aging(Hayflick, L., Exp. Cell Res. 37:611-636 (1965); Norwood, T. H., andSmith, J. R., In: Handbook of the Biology of Aging (2nd ed.) C. E. Finchand E. L. Schneider, eds. Van Nostrand, N.Y. pp. 291-311 (1985);Goldstein, S., Science 249:1129-1133 (1990)). After a limited number ofpopulation doublings, as cells become senescent, they lose thecapability to divide and display a large and flattened morphology. Thecausative mechanisms underlying this phenomenon are not yet understood,despite the many observations that characterize senescent cells at thebiochemical and molecular levels.

One- and two-dimensional protein gel analyses have revealed that thereare few senescent cell-specific marker proteins (Lincoln, D. W. et at.,Exp. Cell Res. 154:136-146 (1984); Wang, E., J. Cell Biol. 100:545-551(1985); Scottie, J. et al., J. Cell Physiol. 131:210-217 (1987);Bayreuther, K. et al., Proc. Natl. Acad. Sci. USA. 85:5112-5116 (1988)).Antigenic determinants that specify senescent cells have been found onthe plasma membrane (Porter, M. B. et al., J. Cell Physiol. 142:425-433(1990)). Components of extracellular matrix, such as fibronectin andcollagenase, have been found to be over-expressed in senescent cells(West, M. D. et al., Exp. Cell Res. 184:138-147 (1989); Kumazaki, T. etal., Exp. Cell Res. 195:13-19 (1991)). However, the relevance of theseobservations to cellular senescence is not clear.

The cell cycle has been found to be regulated and driven by growthfactors. Growth factors act throughout the first gap (G₁) phase of thecell cycle by binding to specific cell surface receptors, which in turntrigger signaling cascades that ultimately govern the transcription ofboth immediate and delayed early response genes. The growth cycle iscontrolled by kinases, especially “cyclin-dependent kinases” (“CDKs”),by the “cyclins” themselves, and by phosphatases (Sherr, C. J., Cell73:1059-1065 (1993), herein incorporated by reference).

Considerable effort has been expended to identify the mammalian kinasesthat are involved in the DNA synthesis cycle. In vertebrate cells, afamily of cyclins has been identified (see, Xiong, Y. et al., Cell71:505-514 (1992)). Gene sequences encoding several of these cyclinshave been isolated (Motokura, T. et al., Nature 350:512-515 (1991);Xiong, Y. et al., Cell 65:691-699 (1991); Lew, D. J. et al., Cell65:1197-1206 (1991); Xiong, Y. et al., Curr. Biol. 1:362-364 (1991);Matsushime, H. et al., Cell 65:701-7139 (1991); Inaba, T. et al.,Genomics 13:565-574 (1992); Xiong, Y. et al., Genomics 13:575-584(1992)).

The D cyclins interact with CDK2, CDK4 and with CDK5, in order toinitiate the growth cycle at the G₁ stage (Matsushime, H. et al., Cell65:701-7139 (1991); Sherr, C. J., Cell 73:1059-1065 (1993)). CyclinE/CDK2 interactions regulate the initiation of S phase (Lew, D. J. etal., Cell 66:1197-1206 (1991); Koff, A. et al., Cell 66:1217-1228(1991)). Cyclin A has been suggested to interact with CDK2 to regulatethe S phase of the growth cycle (Sherr, C. J., Cell 73:1059-1065(1993)). Cyclins A and B are believed to interact with CDC2 to mediatetermination of S phase and initiation of G₂ phase (Norbury, C. et al.,Ann. Rev. Biochem. 61:441-470 (1992); Fang, F. et al., Cell 66:731-742(1991); Walker, D. H. et al., Nature 354:314-317 (1991).

Recently, changes in the expression of several growth regulated geneshave been identified. Expression of c-fos CDC2, cyclins A and B havebeen found to be impaired in senescent cells (Seshadri, T. et al.,Science 247:205-209 (1990)). Similarly, senescent cells evidence aninability to phosphorylate the retinoblastoma protein (Stein, G. H. etal., Science 249:666-669 (1990)). These observations could potentiallyexplain the inability of the cells to enter S phase, since they are alldeteriorative changes of growth promoting gene expression, however, itis not clear whether they are the cause or result of senescence.

One additional change in gene expression that could have a causal rolein senescence is the inhibitor(s) of DNA synthesis produced by senescentbut not young fibroblasts (see, Spiering, A. I. et al., Exper. Cell Res.195:541-545 (1991). Evidence for the existence of the inhibitor(s) wasfirst obtained from heterokaryon experiments in which senescent cellsinhibited initiation of DNA synthesis in young nuclei within theheterokaryon (Norwood, T. H., et al., Proc. Natl. Acad. Sci. USA.71:2231-2234 (1974); Pereira-Smith, O. M., and Smith, J. R., Somat. CellGenet. 8:731-742 (1982)). Studies with cybrids involving senescentcytoplasts and whole young cells lent further support for the presenceof a surface membrane associated protein inhibitor of DNA synthesis insenescent cells (Dresher-Lincoln, C. K., and Smith, J. R., Exp. CellRes. 153:208-217 (1984)). This was directly demonstrated when surfacemembrane enriched preparations from senescent cells or proteinsextracted from the membranes were found to inhibit DNA synthesis whenadded to the culture medium of young cells (Pereira-Smith, O. M. et al.,Exp. Cell Res. 160:297-306 (1985); Stein, G. H., and Atkins, L., Proc.Natl. Acad. Sci. USA. 83:9030-9034 (1986)). Purification of thatinhibitor by biochemical methods has been unsuccessful to date. However,in microinjection experiments, the presence of a high abundance of DNAsynthesis inhibitory messenger RNA has been demonstrated (Lumpkin, C. K.et al., Science 232:393-395 (1986)).

In order to attempt to clone the gene(s) coding for the DNA synthesisinhibitor(s), a functional screening procedure was employed. This methodled to the isolation and identification of three cDNA species thatexhibit DNA synthesis inhibitory activity when introduced into youngcycling cells. These molecules are a preferred class of the moleculesreferred to herein as “senescent cell derived inhibitors” (“SDI”).

Subsequent to the cloning, isolation, sequencing and characterization ofthe SDI molecules of the present invention (see, for example PCTApplication Publication No. US93/12251), other research groups conductedsimilar efforts. Such subsequent efforts have described the SDI-1molecule of the present invention as WAF1, CIP1, PIC1 and p21 (Harper,J. W. et al., Cell 75:805-816 (1993); El-Deiry, W. S. et al., Cell75:817-825 (1993);

Xiong, Y. et al., Nature 366:701-704 (1993); Hunter, T. et al., Cell75:839-841 (1993)).

II. The Cloning of Inducers of Cellular Senescence

In the practice of the present invention, an efficient method for themolecular cloning of the DNA synthesis inhibitory sequences present insenescent human diploid fibroblasts is preferably employed. As is oftenthe case when attempting to clone biologically important genes, it maynot be possible to purify a desired gene responsible for cellularsenescence, even though the activity of its products could be readilydetected.

One method that might be envisioned for identifying such a gene sequencewould be to employ a differential or subtractive screening of asenescent cell derived CDNA library. This method has been used toidentify cDNA molecules that are overexpressed in cells from WernerSyndrome patients (Murano, S. et al., Molec. Cell. Biol. 11:3905-3914(August 1991)). Werner Syndrome is a rare inherited disorder. It ischaracterized by premature aging. The relevance of Werner Syndrome tonatural aging is unknown.

Unfortunately, such screenings would identify a number of genes that,although important for the characterization of senescent cells, wouldnot be primarily responsible for senescence. Furthermore, technicallimitations in cloning full-length cDNA make it difficult to determinethe function of genes cloned by these methods. For these reasons, suchdifferential methods are nether generally suitable, or the mostdesirable method of identifying senescence-related gene sequences.

In contrast, expression screening provides a preferred method foridentifying and isolating such senescence-related gene sequences. Insuch a screening method, the cDNA is cloned directly into a vector thatis capable of expressing the cloned gene in a recipient cell. Therecipient cells can thus be directly screened for any inhibition in DNAsynthesis.

In expression screening, the most important step is the synthesis ofcDNAs. Enzymes should be carefully chosen to be free of impurities. ThecDNA synthesis is preferably repeated several times to ensure thatsatisfactory results (i.e faithful reverse transcription, and fulllength transcript size) will be obtained. Finally, the cDNA products arepreferably size fractionated to eliminate fragmented and prematurelyterminated cDNA products. Double-stranded cDNA products are thenpreferably divided into fractions based on size, i.e., 0.5-2.0, 2.0-4.5,and 4.5-10 kb fractions. The 2-4.5 kb CDNA fraction was used to make theCDNA library on the assumption that many membrane associated proteinshave a relatively high molecular weight. The cDNAs are inserted into asuitable expression vector, preferably pcDSRαΔ, in which the insertedsequences can be transcribed at high levels in young cells.

The most preferred transfection procedure is DEAE dextran-mediatedtransfection, carried out under conditions that allow for transientexpression in a high percentage of young cycling cells. Since thetransfection frequencies could vary from experiment to experiment, thecDNA pool plasmids were transfected along with a marker plasmid, such aspCMVβ (encoding β-galactosidase), and the labeling index was assayed inonly β-galactosidase positive cells. Generally, co-expression oftransfected genes is quite high, since transfection competent cells willaccept multiple plasmids. This simple co-transfection method enabled theevaluation of DNA synthesis in cells expressing exogenous DNA.

The amount of plasmid to be co-transfected can be readily determinedfrom pilot experiments. When the correlation between the transfectionfrequency and the amount of plasmid added is examined using a markerplasmid, maximum efficiency is obtained at a range of 100-500 ng ofplasmid. Taking into account this result, the cDNA library is preferablydivided into small pools in which every pool contained five independentplasmid clones. Then the co-transfection is carried out withapproximately 100 ng of pCMVβ and approximately 400 ng of cDNA plasmid.These parameters were found to maximize the co-expression of cDNA inβ-galactosidase positive cells without decreasing the transfectionfrequency of the marker plasmid.

After the second round of screening, single plasmids which showed stronginhibition of DNA synthesis can be successfully isolated from the poolthat tested positive during the first round screenings (FIGS. 2A, 2B,2C). In FIGS. 2A, 2B and 2C, cDNA pools which showed positive in thefirst round screenings were divided into individual plasmid, andtransfected again. For every cDNA pool (A, B and C in FIGS. 2A, 2B, 2C,respectively), plasmid No. 1 to 5 represents the result of each singleplasmid transfection. In pool B, No. 1 plasmid was found to be only theempty vector. The inhibitory activities of the plasmids are preferablyfurther confirmed by nuclear microinjection experiments. Suchexperiments provide more direct evidence that the isolated plasmidscontain sequences capable of inhibiting DNA synthesis.

III. The Molecules of the Present Invention

The agents of the present invention (collectively referred to as “SDImolecules”) are capable of either inducing the inhibition of DNAsynthesis in active cells, or suppressing such inhibition in senescentor quiescent cells. As such, they may be used for a wide range oftherapies and applications.

The SDI molecules of the present invention include SDI nucelic acidmolecules (e.g., SDI-1 encoding nucleic acid molecules, SDI-1 fragmentencoding molecules, SDI-1 fusion encoding molecules, SDI antisensemolecules, SDI triplex repessor molecules, etc.), SDI protein molecules(i.e. SDI-1, and its fusions and fragments, antibodies to suchmolecules, and protein analogs and mimetics of such molecules), andnon-protein mimetics and analogs of such molecules.

Such molecules may either naturally occurring or non-naturally occuring.A naturally occuring SDI-1 molecule may be purified, such that one ormore molecules that is or may be present in a naturally occuringpreparation containing the molecule has been removed or is present at alower concentration than that at which it would normally be found.

The molecules of the present invention may be either nucleic acids,proteins, carbohydrates, or, more preferably, organic molecules thathave a tertiary structure which resembles or mimics the structure of aSDI protein molecule. The present invention further concerns the use ofbiologically active fragments of molecules, such as SDI nucleic acidmolecules, SDI protein molecules, etc. in lieu of or in addition to anynaturally occurring SDI molecule. As used herein, a molecule is said tobe “biologically active” with respect to cellular proliferation if it iscapable of mediating an affect on the proliferative capacity of arecipient cell. Such biological activity may be a structural attribute,such as the capacity to mediate antisense repression, or the ability tobind at a particular nucleic acid site, or with a particular active siteof a protein, receptor, etc. (or to compete with another molecule forsuch binding) Alternatively, such an attribute may be catalytic, andinvolve the capacity of the biologically active molecule to mediate achemical reaction or response in a recipient cell.

The present invention permits the isolation of all such SDI molecules ina “purified” form. As used herein, an SDI molecule is said to be“purified” if it is present in a preparation that lacks a molecule thatis normally associated with the SDI molecule in its natural state.Proteins, lipids, nucleic acid sequences that do not encode SDImolecules are examples of molecules that are naturally associated withSDI molecules.

A. SDI Nucleic Acid Molecules and Their Oligo-nucleotide orPolynucleotide Fragments

A preferred class of SDI nucleic acid molecules includes the nucleicacid molecules: SDI-1, SDI-2, and SDI-3, and their biologically activefragments. To identify such fragments, the SDI nucleic acid moleculescan be cleaved, as by mechanical methods or more preferably restrictionendonuclease cleavage to thereby generate candidate fragments. Suchfragments can then be provided to cells, and monitored for theircapacity to inhibit DNA synthesis. In one embodiment, gene sequencesthat encode fragments of protein SDI molecules can be administered to arecipient cell.

By administering fragments of nucleic acid SDI molecules (with orwithout linked sequences) it is possible to assess whether a particularfragment of an SDI nucleic acid molecule has biological activity due toits structure. Thus, for example, such candidate SDI molecules could beintroduced into either a normal, immortalized, or tumor cell, and thecapacity of the cell to undergo further proliferation can be monitored.In this manner, sequences that repress cellular proliferation, or inducequiescence can be identified.

Through the use of such methods, nucleic acid molecules that encode theamino terminal half of SDI-1 have been found to exhibit the capacity toconvert immortalized cells or tumor cells to a quiescent state. Morespecifically, nucleic acid molecules that encode SDI-1 amino acidresidues 1-70 have been found to be capable of inducing cellularquiescence. The recognition that residues 1-70 contain a catalyticdomain of SDI-1 indicates that other fragments of the SDI-1 encodingsequence have catalytic activity. Preferred candidate oligonucleotidefragments include nucleic acid molecules that encode SDI-1 amino acidresidues: 5-70, 10-70, 15-70, 20-70, 25-70, 30-70, 35-70 or 40-70.

As discussed below, one aspect of the present invention concerns methodsfor determining the level of SDI-1 mRNA. Nucleic acid molecules that arecapable of hybridizing to an SDI nucleic acid molecules can be used forsuch diagnostic purposes. As used herein, two nucleic acid molecules aresaid to be capable of hybridizing to one another if the two moleculesare capable of forming an anti-parallel, double-stranded nucleic acidstructure. The molecules are said to be “minimally complementary” ifthey can hybridize to one another with sufficient stability to permitthem to remain annealed to one another under at least conventional“low-stringency” conditions. Similarly, the molecules are said to be“complementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another underconventional “high-stringency” conditions. As will be appreciated,complementary molecules need not exhibit “complete complementarity”(i.e. wherein every nucleotide of one of the molecules is complementaryto a nucleotide of the other), but need only be sufficientlycomplementary in sequence to be able to form a stable double-strandedstructure under defined solvent and salt concentrations. A nucleic acidmolecule is said to be the “complement” of another nucleic acid moleculeif they exhibiy complete complementarity. Conventional stringencyconditions are described by Sambrook, J., et al., (In: MolecularCloning, a Laboratory Manual, 2nd Edition, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (1989)), and by Haymes, B. D., et al. (In:Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington,DC (1985)), both herein incorporated by reference). Departures fromcomplete complementarity are therefore permissible, as long as suchdepartures do not completely preclude the capacity of the molecules toform a double-stranded structure.

The nucleic acid molecules that can be used to hybridize to an SDInucleic acid molecule will preferably be shorter than such SDI molecule.Preferred molecules will be completely complementary to an SDI nucleicacid molecule, and will have a length of between about 15 to about 250nucleotides, and most preferably about 15 to about 30 nucleotides.

Such nucleic acid molecules may be obtained using solid phaseoligonucleotide synthetic methods, however, more preferably, suchmolecules will be obtained via the polymerase-mediated,template-dependent extension of a primer molecule that is complementaryto a fragment of an SDI nucleic acid molecule. Such fragments of SDInucleic acid molecules will have a length of between about 15 to about250 nucleotides, and most preferably about 15 to about 30 nucleotides.Such fragments may be DNA or RNA, and may be incorporated into vectors,or be substantially free of other nucleic acid molecules.

B. The Proteins and Polypeptides Encoded by Nucleic Acid SDI Molecules

The present invention further includes the proteins and polypeptidesencoded by the SDI nucleic acid molecules or their oligonucleotidefragments. The sequences of SDI nucleic acid molecules permits one toascribe and identify encoded protein and polypeptide molecules that canbe used either to suppress the inhibition of DNA synthesis associatedwith quiescence and senescence, or to induce such states inproliferating cells. The amino acid sequence of such molecules can bereadily derived from the known relationship between the nucleotidesequence of a nucleic acid molecule, and the amino acid sequence of theprotein it encodes. Therapeutically active proteins and polypeptides canbe identified using a method that is analogous to the above-describedmethod for identifying therapeutically active SDI nucleic acidfragments. By mutating such proteins, it is possible to identifymolecules that have lost the capacity to inhibit DNA synthesis. Amongsuch mutated molecules will be proteins that are capable of exerting adominant effect sufficient to reverse the inhibitory effect ofSDI-encoded proteins and polypeptides.

In one embodiment, such molecules are identified by cleaving nucleicacid SDI molecules, and then incorporating the cleavage fragments intotranslatable expression vectors. In this manner, a library of nucleicacid molecules, each producing a different peptide fragment can beobtained and evaluated. Such expression may be free of additional orextraneous protein, or may comprise a fusion of an SDI fragment to aparticular fusion protein. The biological activity of the expressedproteins can be assessed either by introducing such fragments intorecipient cells, and determining the affect of such introduction onquiescence or proliferation. Alternatively, such molecules can be passedthrough columns that have been pretreated to bind biologically activeSDI molecules. Such columns may, for example, contain bound p53, SDI-1,RB, cyclin D, cdk2, p53- or RB-associated proteins, etc., in order toidentify fragments that have the capacity to bind to such molecules.

Examples of protein fragment SDI molecules include the first 70 aminoacids of SDI-1, which has a biological activity similar to that ofSDI-1. Smaller fragments (such as those containing SDI-1 residues: 5-70,10-70, 15-70, 20-70, 25-70, 30-70, 35-70 or 40-70) may also be employed.Protein fragments that possess SDI-1 amino acid residues 29-45 areparticularly desirable. When conserved amino acid substitutions areconsidered, this region of SDI-1 exhibits 31% identity and 62%similarity to PCNA. Since SDI-1 and PCNA both interact with cyclin-Cdkcomplexes, the conserved region in common (SDI-1 amino acid residues29-45) are believed to be involved in such interactions. Hence,fragments of SDI-1 that contain this conserved region compriseinhibitors of SDI-1, and may indeed exhibit SDI-1 function.

Typically, the SDI proteins and protein fragments will be produced freeof any additional amino acid residues. Alternatively, the SDI proteinsand protein fragments may be produced fused to an amino acid or to apolypeptide. Such synthesis may be accomplished using conventionalpeptide synthetic means, or, more preferably, using recombinant methods.Where fusion molecules are desired, such molecules may containselectable cleavage sites such that the SDI portion of the fusionmolecule may be cleaved from the remaining portion(s) of the fusionprotein.

A particularly preferred fusion molecule results from fusing aglutathione S-transferase glutathione binding sequence to the aminoterminus of the SDI molecule. Such fusion proteins can be readilyrecovered by their retention to a column. The fusion protein can beremoved from the column by washing with glutathione. An GST-SDI-1 fusionprotein can be produced by peptide synsthesis, such as by synthesizingthe SDI protein as a fusion with glutathione S-transferase.Alternatively, recombinant DNA methods can be used to join aGST-encoding polynucleotide to a polynucleotide that encodes SDI-1 or afragment thereof. The sequence of glutathione S-transferase (GST) isknown, and vectors that contain this sequence have been described (see,Ross, V. L., et al., Biochem, J. 294:373-380 (1993); Comstock, K. E. etal., J. Biol. Chem. 268:16958-16965 (1993); Takahasi, Y. et al., J.Biol. Chem. 268:8893-8898 (1993); Klone, A. et al., Biochem. J.285:925-928 (1992); Sternberg, G. et al., Protein Express. Purif.3:80-84 (1992); Morrow, C. S. et al., Gene 75:3-12 (1989)).

An alternative preferred fusion molecule has an amino terminal [His]₆leader sequence. The presence of such leader sequences does notsubstantially reduce the activity of the SDI proteins. More preferably,a leader sequence having the sequence MRGSHHHHHHGA [SEQ ID NO:4] coupledto the amino terminal methionine of SDI-1 will be employed.

A preferred fusion includes essentially all of the GST protein. Anespecially preferred fusion employs the GST of Schistosoma japonicum.The sequence of this GST is described by Smith et al (Gene 67:31(1988)), and the polynucleotide that encodes this GST can be obtainedcommercially (pGEX-2T; Pharmacia). The DNA sequence encoding the GST ofSchistosoma japonicum is shown as SEQ ID NO:5; the encoded amino acidsequence is shown in SEQ ID NO:6. Any of a variety of methods may beused to create such a preferred fusion; a detailed method is provided inExample 20. In one embodiment, a restriction endonuclease recognitionsite in the Schistosoma japonicum GST-encoding polynucleotide can beused to cleave that polynucleotide. The cleavage product can then beligated to a polynucleotide that encodes a desired SDI molecule. Thus,for example, a preferred fusion can be made by cleaving the GST-encodingsequence of Schistosoma japonicum with BamHI so as to obtain apolynucleotide fragment that contains nucleotides 1-673 of theGST-encoding polynucleotide (the BamHI cleaving at a site located atnucleotides 673-678 of the molecule) and then ligating that fragment toan SDI gene sequence (such as a polynucleotide that encodes SDI-1).Where, for example, a polynucleotide that encodes SDI-1 is employed, thegene fusion would link the GST fragment to the SDI-1 polynucleotide suchthat, upon expression, a fusion protein containing the first 226 of the232 amino acids of GST linked to the amino terminus of SDI-1 would beproduced.

As discussed below, one aspect of the present invention concernsantibodies to SDI-1. The above-described proteins and polypeptides canbe used to elicit polyclonal or monoclonal antibodies that can be usedin accordance with the methods of the present invention.

C. Functional Analogs of the SDI Molecules

The present invention also pertains to “functional analogs” of the SDImolecules. Such analogs include both “classical analogs” and “mimeticanalogs.” A classical analog of an SDI molecule is one that has asimilar biological activity, and is chemically related to the SDImolecule. By way of illustration, a non-naturally occurring mutantprotein having SDI activity would comprise a classical analog of aprotein SDI molecule. Similarly, a mutated SDI nucleic acid moleculewould comprise an example of a classical analog of an SDI gene sequence.Likewise, an SDI molecule isolated from a non-human mammalian species(such as a mouse, monkey, etc.) would comprise an example of a classicalanalog of an SDI gene sequence. In contrast, a “mimetic analog” of anSDI molecule retains the biological activity of the molecule, but willtypically be unrelated chemically. An organic molecule whose structuremimics the active site of an SDI protein would comprise a “mimeticanalog” of that protein. Similarly, non-nucleic acid molecules capableof binding to a nucleic acid binding site of SDI, or recognized by SDIwould be a mimetic analog of that molecule.

Thus, functional analogs may be either an oligonucleotide orpolynucleotide, a proteinaceous compound (including both glycosylatedand non-glycosylated proteins), or a non-proteinaceous compound (such asa steroid, a glycolipid, etc.) provided that the agent mimics thefunction of either an entire SDI nucleic acid molecule, or anoligonucleotide or polynucleotide fragment thereof, or a protein orpolypeptide encoded by such a molecule or fragment. Preferred classicalanalogs include polypeptides (including circular as well as linearpeptides) whose sequences comprise the active catalytic or binding sitesof an SDI protein, or oligonucleotide fragments of nucleic acid SDImolecules that are capable of either repressing or inducing SDIactivity. Preferred mimetic analogs include polypeptides that are notfragments of an SDI protein, or mutants thereof, but neverthelessexhibit a capacity to induce quiescence in an SDI-like manner, or toinduce cellular proliferation in the manner of an SDI antagonist.

Classical analogs can be identified either rationally, as describedbelow, or via established methods of mutagenesis (see, for example,Watson, J. D. et al., Molecular Biology of the Gene, Fourth Edition,Benjamin/Cummings, Menlo Park, Calif. (1987). Significantly, a randommutagenesis approach requires no a priori information about the genesequence that is to be mutated. This approach has the advantage that itassesses the desirability of a particular mutant on the basis of itsfunction, and thus does not require an understanding of how or why theresultant mutant protein has adopted a particular conformation. Indeed,the random mutation of target gene sequences has been one approach usedto obtain mutant proteins having desired characteristics (Leatherbarrow,R. J. Prot. Eng. 1:7-16 (1986); Knowles, J. R., Science 236:1252-1258(1987); Shaw, W. V., Biochem. J. 246:1-17 (1987); Gerit, J. A. Chem.Rev. 87:1079-1105 (1987)). Alternatively, where a particular sequencealteration is desired, methods of site-directed mutagenesis can beemployed. Thus, such methods may be used to selectively alter only thoseamino acids of the protein that are believed to be important (Craik, C.S., Science 228:291-297 (1985); Cronin, C. S. et al., Biochem.27:4572-4579 (1988); Wilks, H. M. et al., Science 242:1541-1544 (1988)).

Nucleic acid analogs of SDI molecules can be evaluated by their capacityto be regulated by p53, or other cellular regulators. Alternatively,their capacity to affect cellular proliferation can be directly assayed.For protein analogs of SDI such studies can be accomplished by purifyingthe mutant protein, and comparing its activity to an SDI molecule. Theanalysis of such mutants can also be facilitated through the use of aphage display protein ligand screening system (Lowman, H. B. et al.,Biochem. 30:10832-10838 (1991); Markland, W. et al., Gene 109:13-19(1991); Roberts, B. L. et al., Proc. Natl. Acad. Sci. (U.S.A.)89:2429-2433 (1992); Smith, G. P., Science 228:1315-1317 (1985); Smith,R. P. et al., Science 248:1126-1128 (1990), all herein incorporated byreference)). In general, this method involves expressing a fusionprotein in which the desired protein ligand is fused to the C-terminusof a viral coat protein (such as the M13 Gene III coat protein, or alambda coat protein).

Mimetic analogs of naturally occurring SDI molecules may be obtainedusing the principles of conventional or of rational drug design(Andrews, P. R. et al., In: Proceedings of the Alfred Benzon Symposium,volume 28, pp. 145-165, Munksgaard, Copenhagen (1990); McPherson, A.Eur. J. Biochem. 189:1-24 (1990); Hol, W. G. J. et al., In: MolecularRecognition: Chemical and Biochemical Problems, Roberts, S. M. (ed.);Royal Society of Chemistry; pp. 84-93 (1989); Hol, W. G. J.,Arzneim-Forsch. 39:1016-1018 (1989); Hol, W. G. J., Agnew. Chem. Int.Ed. Engl. 25:767-778 (1986) all herein incorporated by reference).

In accordance with the methods of conventional drug design, the desiredmimetic molecules are obtained by randomly testing molecules whosestructures have an attribute in common with the structure of a “native”SDI molecule, or a molecule that interacts with an SDI molecule. Thequantitative contribution that results from a change in a particulargroup of a binding molecule can be determined by measuring the capacityof competition or cooperativity between the native SDI molecule and theputative mimetic.

In one embodiment of rational drug design, the mimetic is designed toshare an attribute of the most stable three-dimensional conformation ofan SDI molecule. Thus, the mimetic analog of a SDI molecule may bedesigned to possess chemical groups that are oriented in a waysufficient to cause ionic, hydrophobic, or van der Waals interactionsthat are similar to those exhibited by the SDI molecule. In a secondmethod of rational design, the capacity of a particular SDI molecule toundergo conformational “breathing” is exploited. Such “breathing”—thetransient and reversible assumption of a different molecularconformation—is a well appreciated phenomenon, and results fromtemperature, thermodynamic factors, and from the catalytic activity ofthe molecule. Knowledge of the 3-dimensional structure of the SDImolecule facilitates such an evaluation. An evaluation of the naturalconformational changes of an SDI molecule facilitates the recognition ofpotential hinge sites, potential sites at which hydrogen bonding, ionicbonds or van der Waals bonds might form or might be eliminated due tothe breathing of the molecule, etc. Such recognition permits theidentification of the additional conformations that the SDI moleculecould assume, and enables the rational design and production of mimeticanalogs that share such conformations.

The preferred method for performing rational mimetic design employs acomputer system capable of forming a representation of thethree-dimensional structure of the SDI molecule (such as those obtainedusing RIBBON (Priestle, J., J. Mol. Graphics 21:572 (1988)), QUANTA(Polygen), InSite (Biosyn), or Nanovision (American Chemical Society).Such analyses are exemplified by Hol, W. G. J. et al. (In: MolecularRecognition: Chemical and Biochemical Problems, Roberts, S. M. (ed.);Royal Society of Chemistry; pp. 84-93 (1989)), Hol, W. G. J.(Arzneim-Forsch. 39:1016-1018 (1989)), and Hol, W. G. J., Agnew. Chem.Int. Ed. Engl. 25:767-778 (1986)).

In lieu of such direct comparative evaluations of putative SDI analogs,screening assays may be used to identify such molecules. Such an assaywill preferably exploit the capacity of the SDI analog to affectcellular proliferation or quiescence. Alternatively, the molecules maybe applied to a column containing a binding ligand, such as p53, Rb,cyclin D, etc., and the capacity of the molecule to bind to the columnmay be evaluated in comparison to the SDI molecule. Alternatively, amutated SDI molecule (that inhibits the SDI-mediated inhibition of DNAsynthesis) can be administered with a suspected antagonist compound. Thecells would in this case be monitored to determine whether the compoundis able to re-establish an inhibition of DNA synthesis.

Such assays are particularly useful for identifying peptide oroligonucleotide fragments or mimetics of SDI molecules, or analogs ofsuch molecules. Thus, for example, one may incubate cells in thepresence of either an oligonucleotide or a peptide SDI analog (orfragment) and a suspected antagonist compound. The cells would bemonitored in order to determine whether the compound is able to impairthe ability of the SDI oligonucleotide to inhibit DNA synthesis. Asindicated above, column competition assays could alternatively beconducted. Thus, desired SDI classical and mimetic analogs may beidentified by a variety of means.

Significantly, an appreciation of the mechanisms through which SDImolecules mediate their inhibition of DNA synthesis provides analternative, or complimentary, approach to the isolation and recognitionof analogs.

As indicated above, cyclin-dependent kinases play an important role incontrolling the process of cellular DNA synthesis (Draetta, G. et al.,Trends Biol. Sci. 15:378-383 (1990)). The SDI molecules of the presentinvention may be used to dissect the role of such kinases, and theinvolvement of such cyclins, and to thereby identify SDI analogs thatcan be used to inhibit DNA synthesis. For example, the D-type cyclinsare believed to play a role in the G1 or S phase of DNA synthesis(Xiong, Y. et al., Cell 71:505-514 (1992)). The level of cyclin D/cyl1protein increases throughout G1, declines during S and G2, and reaches anadir after mitosis (Matsushime, H. et al., Cell 65:701-7139 (1991);Klyokawa, H. et al., Proc. Natl. Acad. Sci. (U.S.A.) 89:2444-2447(1992); Xiong, Y. et al., Cell 71:505-514 (1992)).

Xiong, Y. et al. (Cell 71:505-514 (1992)) reported the existence of a 21kd polypeptide that associates with cyclin D1 and CDK2. An immunologicalprecipitation method was used in which radiolabelled extracts of cellswere incubated in the presence of anti-cyclin antibodies. Proteins thatassociated with these antibodies were subjected to electrophoresis, andvisualized.

The sequence of this 21 kd polypeptide has now been determined, andfound to be encoded by SDI-1. Thus, since SDI-1 encode molecules inhibitDNA synthesis, the present invention establishes a biological role forthe 21 kd protein (e.g., controlling the transit of the cells from G₁ toS, and from S to G₂. Moreover, since the SDI-1 encoded protein interactswith cyclin D1 and CDK2, the present invention establishes that agentsthat inhibit or reduce this interaction will be analogs of the SDImolecules.

The immunological precipitation method used by Xiong, Y. et al. (Cell71:505-514 (1992)) to demonstrate the association of the 21 kdpolypeptide and the cyclin D1 and CDK2 molecules may be exploited todetermine the mechanism or pathway through which other SDI moleculesmediate their inhibitory effect, and thereby permit the identificationof other analogs.

For example, cells infected with an SDI-2 or SDI-3 sequence, or withantisense molecules for either can be analyzed to determine whether theyexpress any of the proteins that have been previously shown to bind to acyclin molecule. This can most readily be done by determining whether aparticular protein is immunoprecipitated when an extract of treatedcells is incubated with an anti-cyclin antibody. Since such a methodidentifies the molecules with which the SDI molecules interact, itestablishes the pathway through which such SDI molecules mediate theirinhibitory action. Molecules that impair or effect that pathway areanalogs of such SDI molecules. Thus, the methods of the presentinvention can be used to identify inhibitors of any of the regulators ofthe cell cycle. Indeed, the SDI-1 protein has been found to interactwith CDK4 and CDK5 as well as CDK2. Thus, it is likely that the SDI-1protein interacts with multiple CDK and cyclin molecules.

Thus, the recognition that SDI molecules exert their control overcellular proliferation through interactions with cylins and CDKmolecules provides an alternate approach to the identification of SDIanalogs. In a similar manner, the recognition that other cellularregulators mediate their actions by regulating SDI transcription orexpression provides yet another alternate method for identifying SDIanalogs.

For example, the transcription of the SDI-1 gene has been found to beregulated by “tumor suppressor” genes, and most notably by the p53 tumorsuppressor gene. Indeed, the “tumor suppressor” capacity of p53 resultsfrom its capacity to induce SDI-1 expression, and thereby inducecellular quiescence in tumor cells.

The p53 gene has been previously found to encode a tumor-suppressingprotein (Sager, R., Science 246:1406-1412 (1989); Finlay, C., Cell57:1083 (1989); Weinberg, R. A., Scientific Amer., Sept. 1988, pp44-51); Lane, D. et al. (Genes Devel. 4:1-8 (1990)). The p53 gene hasalso been found to play a protective role against the transformingeffects of Friend erythroleukemia virus (Munroe, D. et al. Oncogene2:621 (1988)), and to influence chromosome stability, differentiationand quiescence, and cell proliferation (Sager, R., Science 246:1406-1412(1989)). It has been found that wild type p53 is necessary for cellcycle arrest following ionizing radiation and the constitutiveexpression of wild type p53 can arrest mammalian cells in G1. Theability to induce cell cycle arrest is thought to be related to p53'stumor suppressor function. The protein encoded by the p53 gene is anuclear protein that forms a stable complex with both the SV40 large Tantigen and the adenovirus E1B 55 kd protein.

Approximately 50% of all tumor cells evidence a mutation that diminishesor obliterates p53 expression. The p53 gene has been implicated ashaving a role in colorectal carcinoma (Baker, S. J. et al., Science244:217-221 (1989)). Studies have shown that allelic deletions thatencompass the p53 locus occurred in over 75% of colorectal carcinomas(Baker, S. J. et al., Science 244:217-221 (1989)). The deletion of theregion was found to mark a transition from a (benign) adenocarcinomastage to a (malignant) carcinomatous stage (Vogelstein, B. et al., NewEngl. J. Med. 319:525 (1988)).

Similar deletions in chromosome 17 have been identified in a widevariety of cancers including breast and lung cancers (Mackay, J. et al.,Lancet ii:1384 (1988); James, C. D. et al., Canc. Res. 48:5546 (1988);Yakota, J. et al., Proc. Nat'l. Acad. Sci. (U.S.A.) 84:9252 (1987);Toguchida et al., Canc. Res. 48:3939 (1988)). A variety of human tumors(brain, colon, breast, lung) are characterized by cells that have lostone of the two normal p53 alleles, and have sustained a point mutationin the remaining p53 allele (Nigro et al., Nature 342:705-708 (1989)).Fearon et al. (Cell 61:759-767 (1990)) have hypothesized that both pointmutations and deletions in the p53 alleles may be required for a fullytumorigenic phenotype. These findings suggest that the p53 gene may havea role in many types of cancers.

Recent evidence has suggested that a mutation in the p53 gene may beresponsible for the Li-Fraumeni Syndrome, a rare human genetic disorder(Malkin, D. et al., Science 250:1233-1238 (1990); Marx, J., Science250:1209 (1990), both references herein incorporated by reference).Individuals afflicted with this disease are highly susceptible toseveral malignant tumors-breast carcinomas, soft tissue sarcomas, braintumors, osteosarcomas, leukemia, and adrenocortical carcinoma. Thedisease is also associated with a higher incidence of melanoma, gonadalgerm cell tumors, and carcinomas of the lung, pancreas and prostate (Li,F. P. et al., Ann. Intern. Med. 71:747 (1969); Birch, J. M. et al., J.Clin. Oncol. 8:583 (1990); Birch, J. M. et al., Brit. J. Canc. 49:325(1984); Li, F. P. et al., Canc. Res. 48:5358 (1988); Williams, W. R. etal., Familial Canc., 1st Int. Res. Conf. p. 151 (Karger, Basel, 1985);Strong, L. C. et al., J. Natl. Canc. Inst. 79:1213 (1987)).

Despite the extensive prior characterization of the biological role ofthe p53 gene, the mechanism through which its gene product mediated itstumor suppressor activity had not been previously elucidated. One aspectof the present invention relates to the discovery of that this mechanisminvolves SDI-1. Normal p53 protein increases the expression of SDI-1,and such increased expression suppresses cellular proliferation. Intumor cells that lack p53 function, SDI-1 levels are quite low, thuspermitting cellular proliferation to occur.

The physiological significance of inhibition of cell proliferation byoverexpression of SDI-1 is strengthened by the finding that SDI-1 caninhibit the kinase activity of cyclin/cdk2 complexes. Addition of aGST-SDI-1 fusion protein to cyclin/cdk2 complexes immunoprecipitatedfrom HeLa cell extracts by cdk2 antisera resulted in half maximalinhibition of histone H1 kinase activity.

Thus, molecules that inhibit the tumor suppressor activity of p53 areantagonists of SDI-1; similarly, molecules that enhance the tumorsuppressor activity of p53 protagonists of SDI-1, and are alsoencompassed by the present invention.

In normal human cells, SDI-1 has been found to exist as a complex with acyclin, a CDK, and the proliferating cell nuclear antigen, “PCNA” (Waga,S. et al., Nature 369:574-578 (1994), herein incorporated by reference).PCNA is involved in the excision repair pathway through which cellsrepair damaged DNA. SDI-1 acts to block PCNA's ability to activate DNApolymerase δ. Thus, if cellular DNA is damaged before S phase, theinduction of p53 leads to the transcriptional activation of the SDI-1gene. The expressed SDI-1 protein complexes with CDK/cyclin-mediated DNAreplication, and thus permits PCNA to mediate the excision repair of thedamage. If the cellular DNA is damaged during S phase, excision repairwould lead to increased damage potential. Hence, when damage occursduring S phase, the expressed SDI-1 protein complexes with PCNA to haltthe replication process. As demonstrated below (Example 22), a 23 kDprotein has been identified that exhibits the characteristics of aninactive, phosphorylated derivative of the SDI-1 protein. Thisderivative may be involved in the mechanism through which SDI-1inhibition of PCNA is relieved after the completion of the DNA repairprocess.

Such an understanding of the interrelationship between SDI-1 and PCNAmay be used to identify antagonists or mimetics of SDI-1. Thus, forexample, molecules that inhibit the capacity of SDI-1 to complex withPCNA in cells undergoing S phase comprise antagonists and inhibitors ofSDI-1 action. Conversely, molecules that enhance the capacity of SDI-1to complex with PCNA in cells undergoing S phase comprise SDI-1mimetics.

D. Antagonists of the SDI Molecules

The present invention thus also pertains to antagonists of the SDImolecules. Such antagonists may comprise SDI analogs that compete with,or that inhibit SDI function. Alternatively, such antagonists maycomprise analogs of molecules such as cell cyclins or p53, that interactwith SDI molecules.

Any of a variety of methods can be used to identify polypeptides ornon-proteinaceous molecules that inhibit or repress SDI function. Suchmolecules can be evaluated to determine whether they compete with normalSDI molecules, or whether their presence in a cell affects the capacityof an SDI molecule to induce a quiescent state. For example, nucleicacid molecules that competitively bind p53 molecules are antagonists ofSDI molecules. Such competitors can be readily identified using affinitycolumns, or by DNAse-footprinting methods.

Analogs of p53, or other tumor suppressor proteins, that are capable ofinteracting with and activating SDI sequences are an additional class ofantagonists. Inhibitors of p53 (and other tumor suppressors) arelikewise antagonists of the SDI molecules. Such molecules may beobtained by, for example, mutagenizing p53-encoding cDNA, andidentifying p53 mutants that retain the capacity to bind to SDI-1 genesequences or to SDI-1 proteins, but are otherwise inactive. Thesequences of the cDNA and genomic forms of the p53 gene have beendetermined (Pennica, D. et al., Virol. 134:477-482 (1984); Jenkins, J.et al., Nature 312:651-654 (1984); Oren, M. et al., EMBO J. 2:1633-1639(1983); Zahut-Houri, R. et al., Nature 306:594-597 (1983), all of whichreferences are herein incorporated by reference).

Alternatively, candidate inhibitors can be provided to a recipient celland their capacity to impair normal p53 function can be ascertained. Forexample, such molecules can be tested for their capacity to prevent p53from forming complexes with the SV40 large T antigen (see, DeCaprio, J.A. et al., Cell 54:275-283 (1988); Crawford, L. V., Int. Rev. Exper.Pathol. 25:1-50 (1983)).

Similarly, a variety of means can be exploited in order to identifynucleic acid molecules that inhibit or repress SDI-mediated inhibitionof DNA synthesis. For example, the SDI nucleic acid sequences can bemutated, and the mutated sequences provided to cells in order toidentify cells that do not exhibit an inhibition of DNA synthesis, andwhich have therefore received the desired mutated SDI sequences. In yetanother method, the SDI gene sequences of immortalized cell lines can beevaluated to determine whether they contain mutated SDI genes that havelost the capacity to mediate cellular quiescence. In such manner, it hasbeen determined that some immortalized cells (approximately 10%) carry amutation in the SDI-1 gene that results in the substitution of arginineat amino acid residue 31 of SDI-1 (in place of the serine residuenormally found at this position). Such a finding also implicates residue31 of SDI-1 as being relevant to the active site or conformation ofSDI-1. Since DNA from 12 normal Caucasian donors did not have this SDI-1substitution, it is unlikely that the Arg₃l SDI-1 variant reflects apolymorphism rather than a mutation.

Other mutant SDI proteins have been identified by screening the SDIproteins of various cell lines. Thus, for example, SDI-1 mutants havebeen identified in which the valine normally found at amino acid residue54 has been replaced with alanine, or in which the threonine normallyfound at amino acid residue 80 has been replaced with methionine.

Alternatively, mutated SDI sequences expressed from such nucleic acidmolecules can be evaluated for their capacity to bind p53 protein, orthe gene products of other tumor suppressor genes such as rb, etc.

In yet another embodiment, “triplex” nucleic acid molecules may be usedto provide the desired therapy. A “triplex” molecule is a nucleic acidmolecule that is capable of binding to double-stranded DNA in a mannersufficient to impair its transcription. Such an oligonucleotide can beof any length that is effective for this purpose. Preferably, theoligonucleotide will be about 10-30 nucleotides in length, mostpreferably, about 15-24 nucleotides in length. Triplex oligonucleotidesare disclosed by Hogan, U.S. Pat. No. 5,176,996 and by Varma et al.,U.S. Pat. No. 5,175,266.

The triplex oligonucleotides will preferably be about 20 nucleotides ormore in length, and designed to bind to region of the SDI-1 gene thathas a nucleotide sequence that is either about 2/3 purine or about 2/3pyrimidine. In designing the sequence of the triplex oligonucleotide,the oligonucleotide is constructed to have a G residue when thecomplementary location in the target region is a GC base pair and a Tresidue when the complementary location in the target region is an ATbase pair.

The sequence of the SDI-1 gene may differ from that of the cDNA if thegene contains intervening non-translated sequences (“introns”). In oneembodiment, the genomic SDI-1 sequence is obtained and evaluated for thepresence of regions that comport with the above-described preferredratio of purines to pyrimidines. Such genomic sequences can be obtainedby screening genomic libraries with oligonucleotides probes (e.g., 20-50residues in length) having sequences selected from that of SEQ ID NO:1.Methods for screening genomic libraries are known in the art.

Alternatively, the SDI-1 cDNA sequence can be employed to generatesuitable triplex molecules. An analysis of several hundred genes havingintervening sequences (“introns”) and translated sequences (“exons”) hasrevealed that the intron-exon boundaries have defined upstream anddownstream concensus sequences (Mount, S. M., Nucl. Acids Res.10:459-472 (1982), herein incorporated by reference. The excision of theintrons from mRNA precursor molecules removes most of these concensussequences, and fuses the mRNA to create either a “CAGG” or an “AAGG”exon-exon boundary in the mRNA. Thus suitable target regions of theSDI-1 molecule will preferably (1) be at least 20 nucleotides in length;(2) lack CAGG or AAGG sequences and (3) be either about 2/3 purine orabout 2/3 pyrimidine. Such suitable oligonucleotides can be readilyidentified by mere inspection of SEQ ID NO:1 (e.g., with reference tothe nucleotide positions in SEQ ID NO:1, SEQ ID NO:1₁₋₂₀, SEQ IDNO:1₂₁₋₄₀, SEQ ID NO:1₅₁₋₇₀, SEQ ID NO:1₈₁₋₁₀₀, SEQ ID NO:1₁₂₈₋₁₄₇, SEQID NO:1₁₃₁₋₁₅₀, SEQ ID NO:1₁₅₁₋₁₇₀, SEQ ID NO:1₂₄₁₋₂₆₀, SEQ IDNO:1₂₈₈₋₃₀₇, SEQ ID NO:1₃₀₄₋₃₂₃, SEQ ID NO:1₃₂₉₋₃₄₈, SEQ ID NO:1₃₃₄₋₃₅₃,SEQ ID NO:1₃₆₁₋₃₈₀, SEQ ID NO:1₃₉₀₋₄₀₉, SEQ ID NO:1₄₂₁₋₄₄₀, SEQ IDNO:1₄₉₇₋₅₁₆, SEQ ID NO:1₅₂₅₋₅₄₄, SEQ ID NO:1₅₄₁₋₅₆₀, etc. comprise someof the suitable target sites within the first 600 nucleotides of SEQ IDNO:1).

In yet another embodiment, the sequences of the SDI molecules can beused to define “antisense oligonucleotides” that can repress thetranscription or translation of an SDI gene sequence. In general, an“antisense oligonucleotide” is a nucleic acid (either DNA or RNA) whosesequence is complementary to the sequence of a target mRNA molecule (orits corresponding gene) such that it is capable of binding to, orhybridizing with, the mRNA molecule (or the gene), and thereby impairing(i.e. attenuating or preventing) the translation of the mRNA moleculeinto a gene product. To act as an antisense oligonucleotide, the nucleicacid molecule must be capable of binding to or hybridizing with thatportion of target mRNA molecule (or gene) which mediates the translationof the target mRNA. Thus, antisense molecules of the present inventionare capable of binding to an SDI nucleic acid molecule and inhibitingits activity. Antisense oligonucleotides are disclosed in EuropeanPatent Application Publication Nos. 263,740; 335,451; and 329,882, andin PCT Publication No. W090/00624, all of which references areincorporated herein by reference.

The present invention is particularly concerned with those antisenseoligonucleotides, especially fragments of the SDI-1, SDI-2, or SDI-3genes, which are capable of binding to or hybridizing with mRNA or cDNAmolecules that encode an SDI gene product. Thus, in one embodiment ofthis invention, an antisense oligonucleotide that is designed tospecifically block translation of an SDI mRNA transcript can be used tode-repress the inhibition of DNA synthesis in a recipient quiescentcell.

One manner in which an anti-SDI antisense oligonucleotide may achievethese goals is by having a sequence complementary to that of thetranslation initiation region of an SDI mRNA and of sufficient length tobe able to hybridize to the mRNA transcript of an SDI gene. The size ofsuch an oligomer can be any length that is effective for this purpose.Preferably, the antisense oligonucleotide will be about 10-30nucleotides in length, most preferably, about 15-24 nucleotides inlength.

Alternatively, one may use antisense oligonucleotides that are of alength that is too short to be capable of stably hybridizing to an SDImRNA under physiologic, in vivo conditions. Such an oligonucleotide maybe from about 6-10, or more nucleotides in length. To be used inaccordance with the present invention, such an oligonucleotide ispreferably modified to permit it to bind to a locus of the translationregion of an SDI-encoding mRNA. Examples of such modified moleculesinclude oligonucleotides bound to an antibody (or antibody fragment), orother ligand (such as a divalent crosslinking agent (such as, forexample, trimethylpsoralin, 8-methoxypsoralin, etc.) capable of bindingto a single-stranded SDI mRNA molecules.

In yet another embodiment, SDI-1 antisense molecules may be designedsuch that they hybridize to, and stabilize, an unstable segment of theSDI-1 mRNA. Such molecules would thus enhance the transcription andtranslation of SDI-1 in a cell, and lead to an increased ability toinhibit DNA replication. Such molecules may be used to treat cancer, andother diseases or conditions characterized by hyperproliferation.

An anti-SDI antisense oligonucleotide bound to one reactive group of adivalent crosslinking agent (such as psoralin (for example,trimethylpsoralin, or 8-methoxypsoralin) adduct would be capable ofcrosslinking to an SDI mRNA upon activation with 350-420 nm UV light.Thus, by regulating the intensity of such light (as by varying thewattage of the UV lamp, by increasing the distance between the cells andthe lamp, etc.) one may control the extent of binding between theantisense oligonucleotide and an SDI mRNA of a cell. This, in turn,permits one to control the degree of attenuation of SDI gene expressionin a recipient cell.

In general, the antisense oligomer is prepared in accordance with thenucleotide sequence of an SDI gene, and most preferably in accordancewith the nucleotide sequence of SDI-1 provided in FIGS. 5A-5D. Thesequence of the antisense oligonucleotide may contain one or moreinsertions, substitutions, or deletions of one or more nucleotidesprovided that the resulting oligonucleotide is capable of binding to orhybridizing with the above-described translation locus of either an SDImRNA, cDNA or an SDI gene itself.

Any means known in the art to synthesize the antisense or triplexoligonucleotides of the present invention may be used (Zamechik et al.,Proc. Natl. Acad. Sci. (U.S.A.) 83:4143 (1986); Goodchild et al., Proc.Natl. Acad. Sci. (U.S.A.) 85:5507 (1988); Wickstrom et al., Proc. Natl.Acad. Sci. (U.S.A.) 85:1028; Holt, J. T. et al., Molec. Cell. Biol.8:963 (1988); Gerwirtz, A. M. et al., Science 242:1303 (1988); Anfossi,G., et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:3379 (1989); Becker, D.,et al., EMBO J. 8:3679 (1989); all of which references are incorporatedherein by reference). Automated nucleic acid synthesizers may beemployed for this purpose. In addition, desired nucleotides of anysequence can be obtained from any commercial supplier of such custommolecules.

Most preferably, the antisense or triplex oligonucleotides of thepresent invention may be prepared using solid phase “phosphoramiditesynthesis.” The synthesis is performed with the growing nucleotide chainattached to a solid support derivatized with the nucleotide which willbe the 3′-hydroxyl end of the oligonucleotide. The method involves thecyclical synthesis of DNA using monomer units whose 5′-hydroxyl group isblocked (preferably with a 5′-DMT (dimethoxytrityl) group), and whoseamino groups are blocked with either a benzoyl group (for the aminogroups of cytosine and adenosine) or an isobutyryl group (to protectguanosine). Methods for producing such derivatives are well known in theart.

In yet another embodiment, ribozymes can be employed as inhibitors ofSDI-mediated inhibition. Ribozymes (RNA enzymes) are catalytic RNAsequences (containing no protein) that can cleave RNA target moleculeswith which they hybridize (Cech, T. et al., Cell 27: 487 (1981); Cech,T., Science 236: 1532-1539 (1987); Cech, T. et al., Ann. Rev. Biochem.55: 599-630 (1986); James, W., Antivir. Chem. Chemother. 2: 191-214(1991)). Often the substrate is part of the ribozyme itself.

An artificial ribozyme can be designed to specifically cleave a targetRNA by flanking sequences complementary to the target (Haseloff, J. etal., Nature 334: 585-591 (1988); Cameron, F. et al., Proc. Natl. Acad.Sci. USA 86: 9139-9143 (1989); James, W., Antiviral Chemistry &Chemotherapy 2: 191-214 (1991). The minimum requirement for cleavagewithin the target RNA is the location of a suitable three base sequenceGUC, GUA, or GUU preceding the cleavage site. Artificial ribozymeshaving a characteristic “hammerhead” secondary structure have beendesigned by Haseloff, J. et al. (Nature 334: 585-591 (1988); Jeffries,A. et al., Nucleic Acids Res. 17: 1371-1377 (1989); Gerlach et al. WOPatent Application WO89/05852 (1989); Goodchild, J. et al., Arch.Biochem. Biophys. 284: 386-391 (1991); James, W., Antivir. Chem.Chemother. 2: 191-214 (1991)).

E. Protagonists of the SDI Molecules

The present invention thus also pertains to protagonists of the SDImolecules. As used herein, a “protagonist” of an SDI molecule is amolecule that enhances or increases the biological activity of an SDImolecule.

Since p53 is an inducer of SDI expression, it, or a nucleic acidencoding p53, or biologically active fragments of either, may beprovided to cells in conjunction with an SDI molecule in order to obtainincreased SDI expression.

The present invention also provides SDI protagonists other than thenaturally occurring tumor suppressor proteins. Such protagonists maycomprise SDI analogs or may comprise non-analog molecules that interactwith the cellular molecules that interact with SDI molecules. Thus,mutant forms of the p53 protein having enhanced SDI-activating capacitycomprise one illustrative SDI protagonist. Such molecules may beproduced by mutating the p53 gene, and then selecting muteins thateffect more rapid or more extensive induction of SDI-1 activity than thenormal p53 protein.

Similarly, SDI protagonists can be identified through the use ofscreening assays in which, for example, a candidate molecule is providedto a recipient cell along with an SDI molecule, and the capacity of thecandidate molecule to enhance SDI expression is monitored. Theabove-described methods of rational mimetic design can be used to defineSDI protagonists.

F. Antibodies to SDI Molecules

One aspect of the present invention concerns antibodies to SDI proteinsand protein fragments and the diagnostic and therapeutic uses of suchantibodies.

The above-described SDI proteins and protein fragments may be used toelicit the production of antibodies, single-chain antigen bindingmolecules, or other proteins capable of binding an SDI epitope. Suchantibodies may be polyclonal or monoclonal, and may comprise intactimmunoglobulins, of antigen binding portions of immunoglobulins (such as(F(ab′), F(ab′)₂) fragments, or single-chain immunoglobulins producible,for example, via recombinant means.

Murine monoclonal antibodies are particularly preferred. BALB/c mice arepreferred for this purpose, however, equivalent strains may also beused. The animals are preferably immunized with approximately 25 μg ofaffinity purified SDI protein (or fragment thereof) that has beenemmusified a suitable adjuvant (such as TiterMax adjuvant (Vaxcel,Norcross, Ga.)). Immunization is preferably conducted at twointramuscular sites, one intraperitoneal site, and one subcutaneous siteat the base of the tail. An additional i.v. injection of approximately25 μg of antigen is preferably given in normal saline three weeks later.After approximately 11 days following the second injection, the mice maybe bled and the blood screened for the presence of anti-SDI antibodies.Preferably, a direct binding ELISA is employed for this purpose.

Most preferably, the mouse having the highest antibody titer is given athird i.v. injection of approximately 25 μg of SDI protein or fragment.The splenic leukocytes from this animal may be recovered 3 days later,and are then permitted to fuse, most preferably, using polyethyleneglycol, with cells of a suitable myeloma cell line (such as, forexample, the P3X63Ag8.653 myeloma cell line). Hybridoma cells areselected by culturing the cells under “HAT”(hypoxanthine-aminopterin-thymine) selection for about one week. Theresulting clones may then be screened for their capacity to producemonoclonal antibodies (“mAbs”) to SDI protein, preferably by directELISA.

In one embodiment, anti-SDI-1 monoclonal antibodies are isolated usingthe above-described SDI-1 fusions as immunogens and to facilitatescreening. Thus, for example, a group of mice can be immunized using theGST-SDI-1 fusion protein emulsified in Freund's complete adjuvant(approximately 50 μg of antigen per immunization). At three weekintervals, an identical amount of antigen is emulsified in Freund'sincomplete adjuvant and used to immunize the animals. Ten days followingthe third immunization, serum samples are taken and evaluated for thepresence of antibody. If antibody titers are two low, a fourth boostercan be employed. Polysera capable of binding SDI-1 at 1:5,000 dilutioncan be obtained using this method.

In a preferred procedure for obtaining monoclonal antibodies, thespleens of the above-described immunized mice are removed, disrupted andimmune splenocytes are isolated over a ficoll gradient. The isolatedsplenocytes are fused, using polyethylene glycol with Balb/c-derivedHGPRT (hypoxanthine guanine phosphoribosyl transferase) deficientP3x63xAg8.653 plasmacytoma cells. The fused cells are plated into 96well microtiter plates and screened for hybridoma fusion cells by theircapacity to grow in culture medium supplemented with hypothanthine,aminopterin and thymidine for approximately 2-3 weeks. On average, outof every 10₆ spleen cells subjected to fusion yields a viable hybridoma.A typical spleen yields 5-10 ×10₇ spleen cells.

Hybridoma cells that arise from such incubation are preferably screenedfor their capacity to produce an immunoglobulin that binds to SDI-1. Anindirect ELISA may be used for this purpose. In brief, the supernatantsof hybridomas are incubated in microtiter wells that contain immobilizedGST-SDI-1. After washing, the titer of bound immunoglobulin isdetermined using a goat anti-mouse antibody conjugated to horseradishperoxidase. After additional washing, the amount of immobilized enzymeis determined (for example through the use of a chromogenic substrate).Such screening is performed as quickly as possible after theidentification of the hybridoma in order to ensure that a desired cloneis not overgrown by non-secreting neighbors. Desirably, the fusionplates are screened several times since the rates of hybridoma growthvary. In a preferred sub-embodiment, a different antigenic form of SDI-1may be used to screen the hybridoma. Thus, for example, the splenocytesmay be immunized with the GST-SDI-1 fusion, but the resulting hybridomascan be screened using a [His]₆ fusion, such as that having the leadersequence of SEQ ID NO:4.

As discussed below, such antibody molecules or their fragments may beused for either diagnostic or therapeutic purposes. Where the antibodiesare intended for diagnostic purposes, it may be desirable to derivatizethem, for example with a ligand group (such as biotin) or a detectablemarker group (such as fluorescent group, a radioisotope or an enzyme).

Where the antibodies or their fragments are intended for therapeuticpurposes, it may desirable to “humanize” them in order to attenuate anyimmune reaction. Humanized antibodies may be produced, for example byreplacing an immunogenic portion of an antibody with a corresponding,but non-immunogenic portion (i.e. chimeric antibodies) (Robinson, R. R.et al., PCT Patent Publication PCT/US86/02269; Akira, K. et al.,European Patent Application 184,187; Taniguchi, M., European PatentApplication 171,496; Morrison, S. L. et al., European Patent Application173,494; Neuberger, M. S. etal., PCT Application WO 86/01533; Cabilly,S. etal., European Patent Application 125,023; Better, M. et al.,Science 240:1041-1043 (1988); Liu, A. Y. et al., Proc. Natl. Acad. Sci.USA 84:3439-3443 (1987); Liu, A. Y. et al., J. Immunol. 139:3521-3526(1987); Sun, L. K. et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987);Nishimura, Y. etal., Canc. Res. 47:999-1005 (1987); Wood, C. R. et al.,Nature 314:446-449 (1985)); Shaw et al., J. Natl. Cancer Inst.80:1553-1559 (1988); all of which references are incorporated herein byreference). General reviews of “humanized” chimeric antibodies areprovided by Morrison, S. L. (Science, 229:1202-1207 (1985)) and by Oi,V. T. et al., BioTechniques 4:214 (1986); which references areincorporated herein by reference).

In one therapeutic embodiment, chimeric bivalent antibodies 20 areemployed which contain two different Fab regions, such that the antibodyis capable of binding to an SDI epitope (via the first such Fab region)and to a “non-SDI epitope” (i.e. an epitope of a protein other than anSDI protein) (via the second such Fab region). In one embodiment, such“non-SDI epitopes” are selected such that the chimeric molecule can bindto cellular receptors, such as hormone receptors, immune responsereceptors, etc. Particularly preferred non-SDI receptors includecellular antigens that are indicative of neoplasia, such as antigensassociated with leukemia (Seon et al., Proc. Natl. Acad. Sci., USA80:845 (1983); Aota et al., Cancer Res. 43:1093 (1983); Royston et al.,Transplan. Proc. 13:761 (1981)); colon cancer (Koprowski et al., U.S.Pat. No. 4,349,528; Sakamoto et al., European Patent Publication No.119556; Herlyn et al., Proc. Natl. Acad. Sci., USA 76(3):1438 (1979);Magnani et al., Science 212:55 (1981)); lung cancer (Cuttitta et al.,Proc. Natl. Acad. Sci., USA 78:4591 (1981)); breast cancer (Colcher etal., Proc. Natl. Acad. Sci., USA 78:3199 (1981); Schlom et al., Proc.Natl. Acad. Sci., USA 77:6841 (1980)) and other cancers (See, Lloyd,“Human Tumor Antigens: Detection and Characterization with MonoclonalAntibodies,” In: Herberman, ed., Basic and Clinical Tumor Immunology1:159-214, Nijoff, Boston (1983).

G. Cellular Receptors of SDI Molecules

One aspect of the present invention concerns cellular receptors of SDImolecules, and in particular cellular receptors of SDI-1, forfacilitating the delivery of SDI into target cells.

In one embodiment, such delivery can be accomplished by expressing theSDI molecule as a fusion with a lymphokine, hormone, prohormone, orother molecule that possesses a cellular receptor or a cell-surfaceligand that is capable of binding a receptor. Most preferably, this isaccomplished by ligating a polynucleotide that encodes an SDI molecule(such as SDI-1 cDNA) to a polynucleotide that encodes the protein whichis to recognized and bound by the receptor or cell-surface ligand, andthen expressing the desired fusion protein via recombinant means. Thefusion protein need not contain the complete sequence of the receptorbinding molecule, but may contain only an amount of protein sufficientto permit the desired binding.

In one sub-embodiment, the receptor-binding molecule is selected suchthat the relevant receptor is present on all or most cells. Examples ofsuch molecules include most peptide hormones (such as growth hormone,insulin, etc.) which bind to their respective receptors, transferrinwhich binds to the transferrin receptor, Apo-B protein which binds tothe low density lipoprotein (LDL) receptor, etc. Alternatively, the SDIfusion protein may be selected such that molecule is capable of beingadsorbed by only certain tissue-types or subtypes. Such specificity maybe obtained through the use of molecules that are bound to receptors orligands that are present only on certain populations of cells (such asliver cells, leukocytes, endothelial cells, etc.). Examples of suchmolecules include proteins such as glucagon, gastrin, certain pituitaryhormones (TSH, FSH, etc.), erythropoietin, interleukins,granulocyte-macrophage colony-stimulating factor, neurotrophic proteins,etc. Additionally, proteins capable of binding to cell-surface proteinssuch as CD4, ICAM-1, selectins, ELAMS, LFA-1, etc. may be used.

For example, since ICAM-1 (Simmons, D. et al., Nature 331:624-627(1988); Staunton, D. E. et al., Cell 52:925-933 (1988); Staunton, D. E.et al., Cell 61243-254 (1990) is an endothelial cell-surface ligand forleukocytes that express a CD18/CD11 heterodimer (such as LFA-1, etc.) anICAM-1-SDI fusion molecule would target hematopoietic cells such aslymphocytes. Similarly, a CD4-SDI-1 fusion would be targeted to CD4+ Tcells, and could be used to deliver SDI to such T cells. An LFA-1-SDI-1fusion would target endothelial cells and certain other cell types. Aglucagon-SDI fusion could be used to target liver cells, etc.

In another embodiment, the SDI molecule can be conjugated to anon-protein that can undergo specific binding with a cellular receptor.Examples of such molecules include epinephrine, norepinephrine orhistamine derivatives, prostaglandins, etc.

In yet another embodiment, an endogenous cellular receptor that iscapable of binding an SDI molecule may be expolited to facilitate thedelivery of SDI into a target cell. Such a receptor molecules can beobtained using the above-described SDI proteins and protein fragments.In one method for obtaining the SDI receptor, a DNA (or more preferably,a cDNA) library is produced, preferably from cells that express SDIprotein. The cDNA fragments are cloned into an expression plasmid whichis then introduced into immortalized or tumor cells. The cells are thenincubated in the presence of labeled SDI-1, and evaluated for clonesthat adsorb the SDI-1 to the cell surface, and that exhibit a renewedquiescent or senescent state. Plasmids that encode cellular receptorsare then recovered from such clones.

SDI receptor proteins can also be obtained by expressing cDNA clones inbacteria or other hosts, and then determining whether such clonesproduce proteins that are capable of binding SDI-1. In one preferredsub-embodiment, the cDNA is incorporated into a phage display vector(Lowman, H. B. et al., Biochem. 30:10832-10838 (1991); Markland, W. etal., Gene 109:13-19 (1991); Roberts, B. L. et al., Proc. Natl. Acad.Sci. (U.S.A.) 89:2429-2433 (1992); Smith, G. P., Science 228:1315-1317(1985); Smith, R. P. et al., Science 248:1126-1128 (1990), all hereinincorporated by reference)). As indicated above, this method involvesexpressing a fusion protein in which the desired protein ligand is fusedto the C-terminus of a viral coat protein (such as the M13 Gene III coatprotein, or a lambda coat protein). The phage vectors are then grown toform a library of phage that possess different fusion proteins. Thelibrary is then incubated in the presence of immobilized SDI-1 fusionprotein having a glutathione S-transferase glutathione binding sequenceas its amino terminus. Phage that display cellular receptors of SDI-1are retained by the immobilized SDI-1 fusion protein, and can berecovered by washing the column with glutathione.

In an alternate method of obtaining the SDI receptor, a cellular extractis obtained and incubated in the presence of SDI-1, especially an SDI-1fusion protein having a glutathione S-transferase glutathione bindingsequence as its amino terminus. Proteins that bind to SDI-1 comprisecellular receptor protein.

When desired, the receptor molecule can be solubilized to form a soluble(i.e. not membrane bound) receptor molecule by truncating the proteindomains responsible for anchoring the receptor to the cellular membrane.

IV. Uses of the SDI Molecules of the Present Invention and theirInhibitors

A. Induction of Senescense or Quiescence

Molecules capable of inhibiting SDI function, when provided to arecipient cell cause the immortalization of the cell, and thereby permitthe establishment of a permanent cell line. The antisense, ribozyme andother SDI inhibitor molecules of the present invention may thus be usedto immortalize valuable cell types (such as primary tissue culturecells, etc.) which would otherwise have a transient period ofproliferative viability. They may thus be used for research or to permitor facilitate the accumulation of large numbers of cells, as for organor tissue grafts or transplants. In one embodiment, therefore, theagents of the present invention may be used in conjunction with methodsfor organ or tissue culture to facilitate such methods. Such moleculesmay alternatively be used to effect the immortalization ofimmunoglobulin producing cells, or cells that produce importantbiologicals, such as hormones (insulin, growth hormone, IGF, etc.),immune system modifiers (such as interferons, adhesion molecules,lymphokines, etc.).

Such inhibitory nucleic acid molecules will preferably have nucleotidesequences that are complementary to the sequences of the SDI molecules,and most preferably will be complementary to the sequence of regions orall of the SDI-1 gene. When such oligo- nucleotides are provided torecipient cells, the immortalization of the cell line occurs.Alternatively, the antibodies of the present invention may be used toinhibit SDI activity (e.g., to prevent SDI in a fluid (such as blood)from mediating the quiescence of cells that are in contact with thefluid).

B. Diagnostic Uses

A major use of the molecules of the present invention lies in theircapacity to diagnose the presence and predisposition to cancer. Sincethe absence of SDI-1 expression is the mechanism through whichp53-dependent cancers mediate tumorigenicity, assays of cellular SDI-1expression can be used to diagnose the presence and severity of humancancers. For example, the Li-Fraumeni Syndrome is associated with aparticular set of mutations in exon 7 of the p53 gene (Malkin, D. etal., Science 250:1233-1238 (1990), herein incorporated by reference).Cells of Li-Fraumeni patients do not produce detectable SDI-1 mRNA orSDI-1 protein. Thus, a diagnosis of this disease may be made usinghybridization assays, or immunoprecipitation protocols that measureSDI-1 mRNA or protein levels.

As indicated, approximately 50% of human tumors fail to express normalp53 protein. Thus, assays for p53 activity in biopsy samples is can beused to assess the presence of tumors. Such assays can be readilyaccomplished using the SDI molecules of the present invention,especially the SDI-1 gene sequences, and their fragments. Since p53 isan inducer of SDI expression, the detection of SDI-1 molecules or mRNAin a biopsy material is suggestive of the normal expression of the p53gene.

The anti-SDI antibodies of the present invention may be used in animmunoassay to assess the presence of SDI in a cell, tissue or fluid.Any of a wide array of immunoassays formats may be used for this purpose(Fackrell, J. Clin. Immunoassay 8:213-219 (1985)), Yolken, R. H., Rev.Infect. Dis. 4:35 (1982); Collins, W. P., In: Alternative Immunoassays,John Wiley & Sons, NY (1985); Ngo, T. T. et al., In: Enzyme MediatedImmunoassay, Plenum Press, NY (1985)). The capacity to detect and/ormeasure SDI presence provides a highly desirable means for assessing thepresence or severity of a tumor. Thus, for example, the absence of SDIin a particular tumor indicates that the tumor is more susceptible tometastasis than an SDI-expressing tumor. In one embodiment, theantibodies of the present invention are employed to measure thesolubilized SDI molecules of a sample. The methods of the presentinvention may, however, be used in situ to permit the detection andanalysis of SDI present within a biopsied sample.

The simplest immunoassay involves merely incubating an antibody that iscapable of binding to a predetermined target molecule with a samplesuspected to contain the target molecule. The presence of the targetmolecule is determined by the presence, and proportional to theconcentration, of any antibody bound to the target molecule. In order tofacilitate the separation of target-bound antibody from the unboundantibody initially present, a solid phase is typically employed. Thus,for example the sample can be passively bound to a solid support, and,after incubation with the antibody, the support can be washed to removeany unbound antibody.

In more sophisticated immunoassays, the concentration of the targetmolecule is determined by binding the antibody to a support, and thenpermitting the support to be in contact with a sample suspected ofcontaining the target molecule. Target molecules that have become boundto the immobilized antibody can be detected in any of a variety of ways.For example, the support can be incubated in the presence of a labeled,second antibody that is capable of binding to a second epitope of thetarget molecule. Immobilization of the labeled antibody on the supportthus requires the presence of the target, and is proportional to theconcentration of the target in the sample. In an alternative assay, thetarget is incubated with the sample and with a known amount of labeledtarget. The presence of target molecule in the sample competes with thelabeled target molecules for antibody binding sites. Thus, the amount oflabeled target molecules that are able to bind the antibody is inverselyproportional to the concentration of target molecule in the sample.

In general, immunoassay formats employ either radioactive labels(“RIAs”) or enzyme labels (“ELISAs”). RIAs have the advantages ofsimplicity, sensitivity, and ease of use. Radioactive labels are ofrelatively small atomic dimension, and do not normally affect reactionkinetics. Such assays suffer, however, from the disadvantages that, dueto radioisotopic decay, the reagents have a short shelf-life, requirespecial handling and disposal, and entail the use of complex andexpensive analytical equipment. RIAs are described in LaboratoryTechniques and Biochemistry in Molecular Biology, by Work, T. S., etal., North Holland Publishing Company, NY (1978), with particularreference to the chapter entitled “An Introduction to Radioimmune Assayand Related Techniques” by Chard, T., incorporated by reference herein.

ELISAs have the advantage that they can be conducted using inexpensiveequipment, and with a myriad of different enzymes, such that a largenumber of detection strategies—colorimetric, pH, gas evolution, etc.—canbe used to quantitate the assay. In addition, the enzyme reagents haverelatively long shelf-lives, and lack the risk of radiationcontamination that attends to RIA use. ELISAs are described in ELISA andOther Solid Phase Immunoassays (Kemeny, D. M. et al., Eds.), John Wiley& Sons, NY (1988), incorporated by reference herein.

C. Therapeutic Uses

The molecules of the present invention also posess theraputic utility. Ause is said to be therapeutic if it alters a physiologic condition. Anon-therapeutic use is one which alters the appearance of a user. Theagents of the present invention may be used topically or systemicallyfor a therapeutic or non-therapeutic purpose, such as, for example, tocounter the effects of aging, for example on skin tone, color, texture,etc., or on the degeneration of cells, tissue or organs, such aslymphocytes, vascular tissue (such as arteries, arterioles, capillaries,veins, etc.), liver, kidney, heart and other muscle, bone, spleen, etc.The agents of the present invention may be employed to rejuvenate suchcells, tissue or organs. Thus, they may be used in pharmaceuticals, andthe like, which may comprise, for example, an antisense oligonucleotide,or its equivalent, and a lipophilic carrier or adjunct, preferablydissolved in an appropriate solvent. Such a solvent may be, for example,a water-ethanol mixture (containing 10% to 30% v/v or more ethanol. Suchpreparations may contain 000.1% to 1.0% of the antisenseoligonucleotide. Suitable carriers, adjuncts and solvents are below.

1. Treatment of Cancer and Other Diseases

SDI nucleic acid molecules, their fragments, encoded proteins andpolypeptides, and analogs have use in inducing a senescent or quiescentstate in a recipient cell. Such induction is desirable in the treatmentof age-related disorders (Martin, G. M., Genome 31:390 (1989); Roe, D.A., Clin. Geriatr. Med. 6:319 (1990); Mooradian, A. D., J. Amer. Geriat.Soc. 36:831 (1988); Alpert, J. S., Amer. J. Cardiol. 65:23j (1990));Alzheimer's disease (Terry, R. D., Monogr. Pathol. 32:41 (1990);Costall, B. et al., Pharmacopsychiatry 23:85 (1990)); asthenia andcachexia (Verdery, R. B., Geriatrics 45:26 (1990)), or diseases orconditions in which rapid cellular proliferation is undesirable. In thisrespect, the agents of the present invention can be used therapeuticallyto suppress the rapid proliferation of tumor or tumorigenic cells. Thus,in particular, the molecules of the present invention may be used in thetreatment of cancer, particularly liver, pancreatic, kidney, lung,stomach, breast, uterine, colon, skin, gliomal, lymphatic, prostate,hepatobiliary cancer and malignant melanoma. Indeed, as discussed below,SDI-1 has broad activity in suppressing the proliferation of tumorcells, such as breast, lung, hepatic and glioma tumor lines.

Remarkably, the SDI molecules of the invention have the ability tomediate the differentiation of cancer cells (especially malignantmelanoma cells) into non-cancerous cells.

In one embodiment, such treatment is accomplished by providing SDI-1protein or protein fragments to tumor cells. Such protein may beprovided directly, since SDI-1 appears to be capable of directlyentering tumor cells. Alternatively, SDI-1 may be provided in liposomes,viral sheaths, or other vehicles. In a second embodiment, gene sequencesthat encode SDI-1 or fragments of SDI-1 may be provided as a genetherapy for cancer.

In the case of melanoma or other skin cancers, the SDI molecules of thepresent invention may be provided topically, in an emollient, etc.(preferably formulated with a UV-adsorbing compound, such asp-aminobenzoic acid (PABA).

The SDI molecules of the present invention, particularly when formulatedin a liposomal drug delivery vehicle, can be used to treat bladdercancer. Additional cancer applications for the SDI molecules of theinvention include the inhibition of endothelial cell replication(antiangiogenesis) to prevent neovascularization of tumors, and targetedgene delivery via tumor specific molecules to halt cell growth or induceapoptosis.

The SDI molecules of the present invention may be used alone, or incombination with other conventional chemotherapeutic agents to decreasethe effective concentrations that would otherwise be required in orderto achieve a therapeutic effect.

Indeed, the SDI-1 protein and nucleic acid molecules of the presentinvention have significant utility when used as an adjunct toconventional chemotherapeutic agents. In one embodiment, the SDI-1molecules are administered to tumor cells, thereby enhancing theefficacy of the chemotherapeutic agents. In an alternate orcomplementary embodiment, the SDI-1 molecules are provided (oradditionally provided) sub-sytemically (e.g., locally, or to specificorgans or tissue, or regionally) in order to insulate non-tumor cellsfrom the cytotoxic effects of the chemotherapeutic agents.

The premise of chemotherapy is that cancer cells grow more rapidly thannormal cells, and hence are more sensitive to cytotoxic agents thannormal cells. Each administration of a chemotherapeutic agent kills apercentage of the existing tumor cells, such that multipleadministrations are generally needed in order to completely eliminate atumor (Tenenbaum, L., In: “Cancer Chemotherapy and Biotherapy AReference Guide,” W. B. Saunders Company, Philadelphia, pp. 3-13(1994)).

As indicated above, in one embodiment, the SDI-1 molecules of thepresent invention may be used to increase the sensitivity of cancercells to chemotherapeutic agents, and thus permit the elimination of atumor using lower doses and/or fewer doses of the chemotherapeuticagent/ Such increased sensitivity can be obtained, for example, byproviding a SDI-1 protein (or nucleic acid molecules that express SDI-1)to a patient in concert with the administration of the chemotheraputicagent. Such administration serves to inhibit the replication of thecancer cells (the replication of normal cells is also inhibited by theSDI-1 molecules, however, such inhibition is largely immaterial sincenormal cells proliferate far more slowly than cancer cells). After theSDI-1 molcules are provided to the patient, such administration isterminated or diminished. The effect of such transient or diminishingadministration is to synchonize the cancer cells to the same stage inthe cell cycle (i.e. to freeze the cells, for example, in the G₁ phaseof the cell cycle). Such synchonization significantly increases thesensitivity of the cancer cells to chemotherapeutic agents.

Because such synchronization provides a means for mazimizing thepercentage of cells that are in a particular phase of the cell cycle atthe time of the administration, the administration of SDI-1 moleculesenhances the clinical efficacy of chemotherapeutic agents that exerttheir effect during a specific phase or set of phases of the cell cycle.For example, mitotic inhibitors such as vinca alkaloids (e.g.,vincristine, vinblastine, vindesine, etc.), podophyllum derivatives(e.g., etoposide, teniposide, etc.), taxoids (e.g., taxol, docetaxel,etc.) act during the M phase of the cell cycle by interfering with theformation of the mitotic spindle. Because only a fraction of tumor cellsare in M phase at any given time, such drugs must generally be providedin repeated administrations rather than in a single large dose. Bytreating the cells with SDI-1 molecules, it is possible to synchronizethe tumor cells, such that all (or a large fraction) of the cells willbe at M phase at the same time. Hence, such SDI-1 administration permitsone to employ mitotic inhibitors more effectively.

In a similar manner, antimetbolites such as the folate antagonists(e.g., methotrexate, etc.), purine analogs (e.g., cladribine,fludarabine phosphate, pentostatin, etc.), purine antagonists (e.g.,6-mertcaptopurine, 6-thioguanine, etc.) and pyrimidine antagonists(e.g., 5-fluorouracil, 5-fluorodeoxyuridine, cytarabine, etc.) act oncells in S phase. The efficacy of these agents may be enhanced throughthe adjunct use of SDI-1 molecules.

Such adjunct administration may also be used to improve the selectivityor efficacy of cell cycle phase non-specific chemotherapeutic agents(such as antitumor antibiotics, hydroxyurea, procarbazine, hormones orhormone antagonists, etc.) Despite the observed non-specificity of suchagents relative to the cell cycle phase of tumor cells, it is highlyprobable that synchronization of tumor cell phase will increase theeffectiveness of the chemotherapy.

In accordance with the second embodiment, the SDI-1 molecules of thepresent invention may be used sub-systemically to prevent or toattenuate damage to normal cells. For example, one side effect ofconventional cancer chemotherapy is the damage to rapidly growing tissuesuch as the hair follicles. This damage results in hair loss (alopecia)to affected patients. Despite the transience of such loss, itexacerbates the pyschological trauma, discomfort and depressionassociated with cancer chemotherapy. Such hair loss is thus ofsignificant clinical interest.

The SDI-1 protein or nucleic acid molecules of the present invention maybe used to attenuate or prevent damage or death of hair follicles, andthus provide a treament for hair loss incident to chemotherapy or age.In this embodiment, the SDI-1 molecules would be locally applied (e.g.,topically, transdermally, or intradermally to the scalp, etc.), andpreferably via liposomes. Such administration will inhibit folliculargrowth, and arrest the cells, for example, at G1. The administrationwill thus desensitize the hair follicules to damage caused by asubsequent administration of a conventional chemotherapeutic agent.

The capcity to deliver SDI-1 to hair follicles provides a means fortreating microbial (i.e., fungal, bacterial, or viral) infections of theskin which result from the colonization and infection of the tissue andcells that surround the hair follicles. Examples of such conditionsinclude folliculitis (e.g., boils, carbuncles, etc.), impetigo,fascitis, etc. In particular, SDI-1 (especially when provided inliposomes) can be used to treat acne. Acne results from the colonizationand infection of hair follicles by microorganisms such asProprionobacterium acne, staphylococci, micrococci, and pityrosporumyeasts. The symptoms of acne arise from and are exacerbated by theproduction of sebum by actively proliferating sebaceous cells of thefollicles. The administration of SDI-1 would be convert such activelyproliferating sebaceous cells of the follicle to a quiescent state, andthereby attenuate the production of sebum.

The SDI molecules of the present invention may also be used to treatmucositis (mouth ulcers) that arise as an unavoidable side-effect ofchemotherapy.

The necessity of providing prompt chemotherapy to pregnant cancerpatients jeopardizes their developing fetuses, and hence present medicalpractice must carefully weigh the benefits of early cancer treatmentwith the potential harm that such treatment might cause to the fetus.The SDI-1 protein or nucleic acid molecules of the present invention maybe used to attenuate or prevent damage to developing fetuses in pregnantwomen who must undergo chemotherapy during their pregnancy. The SDI-1protein may be locally provided via injection or infusion into the fetalbloodstream, or into the amnionic fluid which bathes the fetus.Preferably, such local administration is conducted prior to orsimultaneously with the administration of a chemotherapeutic agent tothe mother. The administration of the SDI-1 molecules insulates thefetus from the cytotoxic effects of the chemotherapy.

A large number of non-cancer diseases can also be treated with the SDImolecules of the present invention. The SDI molecules may be used totreat the skin disorder, psoriasis. Particularly when administeredtopically to treat psoriasis, the use of the SDI molecules of thepresent invention can limit the hyperproliferative growth of cellsinvolved in psoriasis with minimal cytotoxic side effects. Rheumatoidarthritis, a debilitating autoimmune disease, may also be treated withthe SDI-1 molecules of the invention.

The SDI molecules of the invention may be used to mediate or acceleratewound healing of both acute wounds (e.g., lacerations, punctures,surgery, etc.) or chronic wounds (e.g., diabetic ulcers, venousulcers,decubitus pressure ulcers).

The molecules of the present invention may be used to treat restenosisincident to angioplasty. The term “stenosis” denotes a narrowing orconstriction of a duct or canal. A variety of disease processes, such asatherosclerotic lesions, immunological reactions, congenitaldeformities, etc., can lead to the stenosis of coronary arteries andthus to myocardial ischemia. Percutaneous transluminal coronaryangioplasty (PTCA), the insertion and partial inflation of a ballooncatheter into a stenotic vessel to effect its repair, has beenextensively used to treat stenosis. The major limitation of PTCA is“restenosis” (i.e. the re-constriction) of the vascular lesion (Liu, M.W. et al., Circulation 79:1374-1386 (1989), herein incorporated byreference). Restenosis has been found to occur in 30% to 40% ofangioplasty patients within 6 months of the procedure (Califf, R. M. etal., J. Amer Col. Cardiol. 17:2B-13B (1991), McBride, W. et al., N.Engl. J. Med. 318:1734-1737 (1988)). Restenosis develops so rapidly thatit may be considered a form of accelerated atherosclerosis induced byinjury. The significance of the high restenosis rate is compounded bythe present inability to predict with a high degree of certainty whichpatients, vessels, or lesions will undergo restenosis. Indeed, arteriesthat are widely patent 2 days after PTCA, free of obstructive thrombus,have exhibited restenosis at catheterization 4-6 months later (Liu, M.W. et al., Circulation 79:1374-1386 (1989)).

Glaucomas can also be treated with the SDI molecules of the presentinvention. “Glaucomas” are a family of debilitating eye diseases thatare each characterized by a progressive loss of visual field (i.e. thesolid angle of vision that defines whether an object is within view).Unless checked, the impairment of the visual field leads to absolute andirreversible blindness.

Glaucomas are characterized by a disruption in the normal flow of the“aqueous” (i.e., the clear fluid of the eye having a composition similarto that of plasma) through the posterior and anterior chambers of theeye (Vaughan, D. et al., In: General Ophthamology, Appleton & Lange,Norwalk, Conn., pp. 213-230 (1992)). The aqueous is produced by theciliary processes and secreted into the posterior ocular chamber. Innormal eyes, it then passes through the pupil and into the anteriorchamber of the eye. The aqueous flows out of the anterior chamber into acollagen-elastin filtering structure known as the “trabecular meshwork,”and ultimately through the “canal of Schlemm” into the venous bloodsupply. The ability of aqueous to traverse the canal of Schlemm dependsupon the presence and extent of transcellular channels and the rate ofoutflow determines the intra-ocular pressure. The average pressure innormal eyes is about 14 mm Hg. In glaucoma, the outflow of aqueous isdisrupted, and intra-ocular pressure increases. Damage usually begins atabout mm Hg, and the eye ruptures at about 240 times average pressurevalues.

The antimitotic agent, 5-fluorouracil, has been proposed as atherapeutic for glaucoma (Sarfarazi, F., U.S. Pat. No. 5,304,561).Unfortunately, 5-fluorouracil is associated with significant adverseside effects. Since the agents of the present invention can preventcellular proliferation, they may be used to inhibit the proliferation ofthe cells of the trabecular meshwork, and accordingly provide a therapyfor glaucoma, especially, primary open angle glaucoma. For such uses, itis desirable to employ SDI-1 molecules that can be administered byintra-ocular means (such as by eye drops, or ointments). The ability ofa drug to traverse the cornea is enhanced if the drug has bothlipophilic and hydrophilic regions. Thus, for intra-ocular delivery, itis desirable to modify the SDI-1 molecules of the invention such thatthey contain such regions. Suitable lipophilic and hydrophilic groupsare known in the art (see, Remington's Pharmaceutical Sciences), andcomprise aliphatic groups, lipids, etc. (lipophilic groups) and organicacids, esters, ionic groups, etc. (hydrophilic groups). Such groups canbe readily added to the SDI-1 molecules of the present invention by, forexample, derivatizing the side chain groups of appropriate amino acids.

Cysteinyl residues may be reacted with α-haloacetates (and correspondingamines), such as chloroacetic acid or chloroacet-amide, to givecarboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues alsoare derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues may be derivatized by reaction withdiethylprocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1 M sodium cacodylateat pH 6.0.

Lysinyl and amino terminal residues may be reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing a-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylissurea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues may be modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK_(a) of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

Carboxyl side groups (aspartyl or glutamyl) may be selectively modifiedby reaction with carbodiimides (R′—N—C—N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3 (4azonia 4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl andglutamyl residues may be converted to asparaginyl and glutaminylresidues by reaction with ammonium ions.

2. Antiviral and Antimicrobial Uses

In an alternative embodiment, the molecules of the present invention mayby used as an anti-viral agent to impair the propagation of visursessuch as influenza, hepatitis, (e.g., hepatitis B or hepatitis C),Epstein-Barr, rhinovirus, pappilomavirus, papovavirus, etc. Inparticular, since SDI molecules (especially, SDI-1 and its analogs) actto inhibit cellular proliferation, and since retroviruses preferentiallyproliferate only in actively dividing cells, the present inventionprovides an antiviral therapy against HIV, and thus can be used to treatdiseases such as AIDS and ARC. Similarly, for conditions such as warts(including venereal warts), larygeal pappilomatosis, progressivemultifocal leucoencephalopathy, etc., the administration of such SDImolecules inhibits the proliferation of infected cells, and thusprovides symptomatic treatment for the condition.

In another embodiment, the molecules of the present invention may beused as an anti-parasitic agent to treat fungal, yeast, protozoan,helminthic, nematodal and other parasitic infections (e.g.,candidiassis, aspergillosis, coccidiomycosis, leishmaniasis, amoebiasis,trichomoniasis, tinea (pedis, crusis, etc.) vaginal monolysis,schistosomiasis and malaria).

In a manner similar to that described above, the administration of SDI-1molecules can increase the therapeutic efficacy of antiviral orantimicrobial agents. Because SDI-1 molecules inhibit the replication ofpathogens, it may be applied topically to skin, or systemically viainjection, in order to prevent or attenuate the risk of subsequentinfection. Thus, for example, the molecules could be incorporated into asuitable pharmaceutical composition, and applied topically to the handsof surgeons or other medical practitioners prior to their exposure topotentially infected individuals.

In another embodiment, the SDI-1 molecules could be administered as anadjunct to antiviral agents in the treatment of suspected or actualviral infection in order to synchronize the cell cycles of any virallyinfected cells, and thereby enhance the therapeutic efficacy of theantiviral agent. Likewise, the SDI-1 molecules could be administered asan adjunct to antiparasitic agents in the treatment of suspected oractual parasitic infection in order to synchronize the cell cycles ofthe cells of the parasite, and thereby enhance the therapeutic efficacyof the antiparasitic agent. Such adjunct administration may be used totreat conditions such as the above-described fungal, yeast, protozoan,helminthic, nematodal or other parasitic infections.

3. Other Therapeutic Uses

The antisense and other SDI inhibitor molecules of the present inventionmay be used to stimulate the proliferation of spermatocytes, or thematuration of oocytes in humans or animals, and thus, may be used toincrease the fertility of a recipient. Conversely, SDI molecules andtheir analogs can be used to inhibit gametogenesis in males or females,and thus can be used as contraceptive agents to induce infertility inmales or females. Such use also provides the benefit of attenuating thereplication and proliferation of virally (e.g., HIV, etc.) infectedcells, and hence serves to lessen the probability of contracting viraldiseases (e.g., AIDS, etc.).

Since the SDI-1 molecules of the present invention are capable ofinhibiting DNA replication, they may be used to prevent or attenuateUV-light induced DNA damage (such as that encountered from overexposureto the sun). In individuals who lack normal capacity to repair suchdamage, the SDI-1-mediated inhibition of DNA synthesis would providegreater opportunity for repair to occur. Hence, the SDI-1 molecules ofthe present invention may be used to treat indivduals suffering fromdeficiencies in DNA repair capacity (e.g., indivduals having xerodermapigmentosum, ataxia telangiectasia, etc.).

Since the antisense and other inhibitor molecules of the presentinvention are capable of stimulating cellular proliferation, they may beused to promote wound healing, angiogenesis, endothelial cellproliferation, recovery from burns, or after surgery, or to restoreatrophied tissue, etc. The antibodies of the present invention may alsobe used to effect wound healing, burn recovery, or subsequent to traumaor surgery. Indeed, all such compounds can also be used to suppressgeneral tissue regeneration or vascularization. For such an embodiment,these agents may be formulated with antibiotics, anti-fungal agents, orthe like, for topical or systemic administration.

The molecules of the present invention may be used to provide genetherapy for recipient patients. In one embodiment, cells or tissue froma patient may be removed from the patient and treated with a molecule ofthe present invention under conditions sufficient to permit arestoration of an active growing state. In one preferred embodiment ofthis use, lymphocytes of an individual (such as, for example, an immunecompromised individual, such as an AIDS patient, etc., or animmune-competent individual who will serve as a donor of lymphocytes)can be removed and treated with antisense SDI nucleic acids. Theadministration of these molecules will derepress the lymphocytes. Afteradministration, the lymphocytes are reintroduced into the patient, andhave an enhanced ability to combat infection.

In yet another embodiment of the present invention, the molecules of thepresent invention can be used to facilitate autologous cell replacement.In this embodiment, the SDI nucleic acid molecules, their fragments,encoded proteins and polypeptides, and analogs can be used to permit thein vitro proliferation of cells (such as bone marrow cells, epithelialcells, muscle cells, hepatic cells, etc.) in order to replenish oraugment the amount or concentration of such cells in a patient. Thus,for example, bone marrow cells can be removed, treated with suchmolecules, and then cultured in vitro until a sufficient mass of cellshas been obtained to augment a desired immune response. Alternatively,hepatic cells (such as hepatic cells that are free of a hepatitis virus)can be removed from a patient, treated, cultured and then transplantedback into the patient in order to treat hepatic disease.

In one sub-embodiment, such treated cells may be themselves directlytransplanted back into the patient, and thus propagate in vivo.Alternatively, as indicated, such cells may be cultured in vitro, andreintroduced when a desired titer has been attained.

In accordance with the above-described embodiments and sub-embodimentsof gene therapy, the SDI-1 cDNA and antisense sequences may be operablylinked to tumor-specific or tissue- specific promoters in order toconfine the therapeutic effect to a desired site or tissue. Examples ofsuitable tumor-specific promoters include those that direct thetranscription of tumor specific antigens such as α-fetoprotein,carcinoembryonic antigen, amylase, γ-glutamyl transferase, etc. Examplesof suitable tissue-specific promoters include the phenylalaninehydroxylase promoter (Wang, Y. et al., J. Biol. Chem. 269:9137-9146(1994); Svensson, E. et al., Eur. J. Hum. Genet. 1:306-313 (1993);Konencki, D. S. et al., Biochemistry 31:8363-8368 (1992)), thealpha-1-antitrypsin (AAT) promoter (Li, Y. et al., Molec. Cell. Biol.8:4362-4369 (1988); the muscle actin promoter, etc. Of particularinterest are promoters that direct expression in breast, liver, lung orcolon tissues.

The molecules of the present invention are particularly suitable for usein the creation and/or study of animal models for disease or tissuedegeneration. Thus, the molecules of the present invention can be usedto study effectors of an animal model that is characterized by abnormalaging or cellular degeneration. Similarly, the administration of the SDImolecules (linked, for example to suitable regulatory sequences in orderto permit their expression in a recipient cell) can be used to createanimal models of aging or of tissue degeneration.

D. Delivery of Pharmacological Agents The capacity of SDI-1 to directlyenter a cell provides a means for accomplishing the delivery ofpharmacological agents to a recipient cell. Thus, in one embodiment, apharmacological agent will be conjugated to SDI-1 and provided to arecipient patient. The presence of the SDI-1 moiety (which may be theintact SDI-1 protein, or a transport-sufficient fragment of SDI-1)transports the attached pharmacological agent into the recipient cell.

Any of a variety of cross-linking agents may be used to conjugate thepharmacological agent to the SDI-1 moiety. Alternatively, such agentscan be provided as fusion proteins (exemplified by the above-discussedGST-SDI-1 fusion proteins). Such fusion proteins can be prepared by, forexample, expressing a nucleic acid molecule that encodes such a fusion.

The pharmacological agents that may be administered to cells in thismanner may include agents that provide a therapeutic benefit to cells(such as an antihypertensive, anti-inflammatory, anti-arrthymic, etc.).Alternatively, the pharmacological agent may adversely affect recipientcells (such as by comprising a cytokine, a toxin, etc.).

Where it is desired that the pharmacological agents to be delivered isSDI-1, such can be accomplished using the anti-SDI antibodies of thepresent invention may comprise chimeric binding regions. By selectingthe binding portions of such chimeric antibodies to include a bindingdomain that is specific for a tumor antigen, it is possible to producean antibody that can bind to both a tumor antigen and to an SDImolecule. Such a molecule can be used to “ferry” an SDI protein into anycell that arrays the tumor antigen. The conveyed SDI molecule can inducethe tumor cell to resume a quiescent or senescent state. Such chimericantibodies thus comprise an anti-cancer treatment.

Alternatively, the SDI molecules of the present invention may beprovided to cells by fusing the SDI molecule to a hormone, or othermolecule that can bind to a desired subset of recipient cells. Thus, forexample by conjugating SDI-1 to a soluble CD4 molecule, or to an insulinmolecule, one coud target the administration of SDI-1 to leukocytes thatarray CD4, or to cells that array the insulin receptor. Although suchconjugates can be produced using a variety of methods, it is preferredto produce such conjugates by expressing nucleic acid molecules thatencode the fusion proteins.

As indicated, a GST-SDI fusion is a particularly preferred fusion. TheN-terminal domain of rat GST exhibits significant homology to humanmigration inhibition factor (MIF) (David, J. R., Parisitology Today9:315-316 (1993); Mikayama, T. et al., Proc. Natl. Acad. Sci. (U.S.A.)90:10056-10060 (1993), herein incorporated by reference). Thus, distinctfrom any action of SDI, GST thus has the potential for binding tocellular receptor that are capable of binding MIF and related molecules.Indeed, one aspect of the present invention involves the recognitionthat a GST-pharmacological agent fusion protein can bind to cellularreceptors and effect the delivery of the pharmacological agent to thetarget cell. Thus, molecules other than SDI could be fused to GSTsequences (such as those described above) in order to effect theirdelivery into a desired target cell.

E. Preparatory Uses

The anti-SDI antibodies of the present invention provide a facile meansfor purifying and recovering SDI protein from solution. In thisembodiment, cellular lysates or extracts are incubated in the presenceof an anti-SDI antibody, preferably immobilized to a solid support. TheSDI molecules bind to the antibody, and can thus be recovered inpurified form.

F. Uses of the SDI Cellular Receptor and Its Solubilized Derivatives

The SDI cellular receptor and its solubilized derivatives can be used tofacilitate the recovery of SDI from SDI protein- containingpreparations. In this manner, the receptor may be used as apseudo-antibody. The receptor may also be used therapeutically tomodulate cellular expression and responses to SDI protein. Thus,receptor molecules can be provided to tumor cells (via liposomes, or byproviding such cells with nucleic acid that encodes the receptor). Suchmolecules will increase the capacity of the tumor cells to respond toSDI presence, and will thereby ameliorate the cancer.

V. Methods of Administration

The SDI molecules of the present invention can be formulated accordingto known methods to prepare pharmaceutically useful compositions,whereby these materials, or their functional derivatives, having thedesired degree of purity are combined in admixture with aphysiologically acceptable carrier, excipient, or stabilizer. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed. The “SDI molecule” of such compositions may be SDI-1 protein,fusions (e.g., GST-fusions, etc.) or fragments of SDI-1 protein ormimetics of such molecules. The SDI molecules may be sense, antisense ortriplex oligonucleotides of the SDI-1 cDNA or gene. A composition issaid to be “pharmacologically acceptable” if its administration can betolerated by a recipient patient. Such an agent is said to beadministered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient patient. In some instances, the presence of anintervening sequence upstream of a protein-encoding nucleic acidsequence can enhance the transcription or expression of specifiedpolynucleotides (Brinster, R. L. et al., Proc. Natl. Acad. Sci. (U.S.A.)85:836-840 (1988); Palmiter, R. D. et al., Proc. Natl. Acad. Sci.(U.S.A.) 88:478-482 (1991); Huang, M. T. F. et al., Nucl. Acids Res.18:937-947 (1990); GRuss et al., Proc. Natl. Acad Sci. (U.S.A.) 76:4317(1979); Hamer, D. et al., Cell 18:1299 (1979); Gasser, et al., Proc.Natl. Acad. Sci. (U.S.A.) 79:6522 (1982); Calles, et al. , Genes &Devel. 1:1183 (1987)). Where the SDI molecules that are to beadministered comprise nucleic acid molecules, such as SDI-1-encodingmolecules or SDI-1 antisense sequences, it is particularly preferred toemploy nucleic acid molecule that include at least one non-translatedintervening sequence within, or adjacent to the relevant SDI sequence.Such use is particularly desirable in transfections of cells in culture,when producing transgenic animals, or when mediating genetic therapy.Since expression is obtained in the absence of such interveningsequences, the relative position or number of intervening sequences isnot critical to the invention. The presence of such a non-translatedintervening sequence can, however, increase the extent of transcriptionand/or expression of SDI nucleic acid molecules. A preferred constructcontains the SDI sequences adjacent to, but not interrupted by, theintervening sequences. An example of a suitable vector is pOPRSVICAT(Stratagene), which contains Rous Sarcoma Virus LTR (long terminalrepeat) promoter that is operably linked (i.e., capable of transcribing)nucleic acid sequences that encode a chloramphenicol acetyl transferase(CAT). An intervening untranslated sequence from SV40 is present betweenthe promoter and the CAT-encoding gene sequences.

Suitable vehicles and their formulation, inclusive of other humanproteins, e.g., human serum albumin, are described, for example, inRemington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack,Easton P A (1980)). In order to form a pharmaceutically acceptablecomposition suitable for storage or administration, such compositionswill contain an effective amount of one or more “SDI molecule.”

If the composition is to be water soluble, it may be formulated in abuffer such as phosphate or other organic acid salt preferably at a pHof about 7 to 8. If the composition is only partially soluble in water,it may be prepared as a microemulsion by formulating it with a nonionicsurfactant such as Tween, Pluronics, or PEG, e.g., Tween 80, in anamount of, for example, 0.04-0.05% (w/v), to increase its solubility.The term “water soluble” as applied to the polysaccharides andpolyethylene glycols is meant to include colloidal solutions anddispersions. In general, the solubility of the cellulose derivatives isdetermined by the degree of substitution of ether groups, and thestabilizing derivatives useful herein should have a sufficient quantityof such ether groups per anhydroglucose unit in the cellulose chain torender the derivatives water soluble. A degree of ether substitution ofat least 0.35 ether groups per anhydroglucose unit is generallysufficient. Additionally, the cellulose derivatives may be in the formof alkali metal salts, for example, the Li, Na, K, or Cs salts.

Optionally other ingredients may be added such as antioxidants, e.g.,ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, mannose,or dextrins; chelating agents such as EDTA; and sugar alcohols such asmannitol or sorbitol.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled or sustained release preparations may beachieved through the use of polymers to complex or absorb the SDImolecule(s) of the composition. The controlled delivery may be exercisedby selecting appropriate macromolecules (for example polyesters,polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate,methylcellulose, carboxymethylcellulose, or protamine, sulfate) and theconcentration of macromolecules as well as the methods of incorporationin order to control release.

Sustained release formulations may also be prepared, and include theformation of microcapsular particles and implantable articles. Forpreparing sustained-release compositions, the SDI molecule(s) of thecomposition is preferably incorporated into a biodegradable matrix ormicrocapsule. A suitable material for this purpose is a polylactide,although other polymers of poly-(α-hydroxycarboxylic acids), such aspoly-D-(−)-3-hydroxybutyric acid (EP 133,988A), can be used. Otherbiodegradable polymers include poly(lactones), poly(orthoesters),polyamino acids, hydro-gels, or poly(orthocarbonates) poly(acetals). Thepolymeric material may also comprise polyesters, poly(lactic acid) orethylene vinylacetate copolymers. For examples of sustained releasecompositions, see U.S. Pat. No. 3,773,919, EP 58,481A, U.S. Pat No.3,887,699, EP 158,277A, Canadian Patent No. 1176565, U. Sidman et al.,“Biopolymers” 22:547 [1983], and R. Langer et al, “Chem. Tech.” 12:98[1982].

Alternatively, instead of incorporating the SDI molecule(s) of thecomposition into polymeric particles, it is possible to entrap thesematerials in microcapsules prepared, for example, by coacervationtechniques or by interfacial polymerization, for example,hydroxymethylcellulose or gelatine-microcapsules andpoly(methylmethacylate) microcapsules, respectively, or in colloidaldrug delivery systems, for example, liposomes, albumin microspheres,microemulsions, nanoparticles, and nanocapsules or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences(1980).

Liposomes are a particularly preferred means for accomplishing thedelivery of SDI (protein or nucleic acid or other) molecules. Such adelivery means is particularly preferred when administering SDImolecules to skin as by topical administration. Although a wide varietyof liposome compositions can be employed, a preferred liposomecomposition is composed of a mixture of positively charged and neutrallipids, such as those disclosed by Eppstein, D. A. et al. (U.S. Pat. No.4,897,355), herein incorporated by reference. An alternative preferredliposome composition is described by Yarosh, D. B. (U.S. Pat. No.5,190,762, herein incorporated by reference), and in particular thepH-sensitive liposomes discussed therein. Such sensitivity causes theliposomes to destabilize at a pH of less 4.5 Such sensitivity isproduced by using phospholipids (such as phosphatidylethanolamine) whichform lipid bilayers when charged, but fail to stack in an orderedfashion when neutralized. The net charge of such phospholipids can bemaintained at a pH which would otherwise neutralize the phospholipid'shead groups by including charged molecules in the lipid bilayer whichthemselves can become neutralized. Examples of suitable chargedmolecules include oleic acid and cholesteryl hemisuccinate (CHEMS) (U.S.Pat. No. 5,190,762).

A particularly preferred liposome formulation contains a 3:1 (w/w)mixture of the polycationic lipid2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium-trifluoroacetate (DOSPA) [Chemical Abstracts designation:N-[2-({2,5-bis(3-aminopropyl)amino-1-oxypentyl}amino)ethyl]-N-,N-dimethyl-2,3-bis(9-octadecenyloxy)-1-propanaminium-trifluoro-acetate, and the neutral lipiddioleolyphosphatidylethanolamine (DOPE) in water. A particularlypreferred liposome having such a composition is LipofectamineTm Reagent(Life Technologies, Inc., Gaithersburg, Md.). The positively charged andneutral lipids form liposomes that can complex with either acidicprotein or nucleic acids (see, Lin, M. F. et al., Biochem. Biophys. Res.Commun. 192:413-419 (1993); Wizel, B. et al., Eur. J. Immunol.24:1487-1495 (1994)). The capacity of such liposomes to deliver a basicprotein such as SDI-1 (predicted pl=8.4) is quite unexpected.

“Transferosomes” are also preferred liposomes for the purposes of thepresent invention. Methods for producing and using transferosomes areprovided by Planas, M. E. et al. (Anesth. Analg. 75:615-621 (1992)),Cevc, G. et al. (Biochim Biophys. Acta 20 1104:226-232 (1992)), Blume,et al. (Biochim Biophys. Acta 1146:157-168 (1993)), Blume, et al.(Biochim Biophys. Acta 1149:180-184 (1993)), all herein incorporated byreference.

Another preferred liposome composition is provided by Weiner, N. D. (PCTApplication WO 91/01719), Egbaria, K. et al. (Antimicrob. AgentsChemother. 33:1217-1221 (1989)), Egbaria, K. et al. (Antimicrob. AgentsChemother. 34:107-110 (1990)), all herein incorporated by reference.

Additionally preferred liposome formulations are disclosed by Handjani,R. M. et al. (U.S. Pat. No.4,830,857), Hope, M. J. et al. (U.S. Pat.Nos. 5,204,112; 5,252,263),and by Vanlerberghe, G., et al. (U.S. Pat.Nos. 5,164,488; 4,827,003; 5,008,406; 4,247,411), all hereinincorporated by reference.

In one embodiment, particularly performed using the above-describedpreferred liposome compositions, preformed liposomes are incubated withSDI protein molecules. Without limitation to the invention, the proteinis believed to unexpectedly adsorb to (or dissolve into) the externalsurface of the liposome, and to thus become arrayed on or in theliposome surface. The protein may traverse the surface of the liposome.

The liposome formulations can be administered to cells in culture (so asto achieve immortalization, or to induce the cessation ofproliferation), or, therapeutically, to treat disease or hyper- orhypo-proliferative conditions.

In one embodiment, such formulations will contain an SDI-1 molecule(e.g., a GST-SDI-1 fusion, etc.) that can mediate its own intracellularuptake. Suitable methods are known in the art, see, for example, forexample, by Biessen, E. A. L. et al. (PCT Application WO94/04545),Felgner, P. L. (PCT Patent Application WO91/17424), Akiyama, K. et al.(PCT Application WO93/20801), Blum, A. et al. (PCT ApplicationWO93/04672), Abai, A. M. et al. (PCT Application WO93/03709), Hosokawa,S. et al. (U.S. Pat. No. 5,264,221), Cullis, P. R. et al. (U.S. Pat.Nos. 5,204,112; 5,252,263), Japanese Patent No. 4,082,893, Phillips, W.T. et al. (U.S. Pat. No. 5,158,760), Weiner, N. D. (PCT ApplicationWO91/01719), Hostetler, K. Y. et al. (U.S. Pat. No. 5,223263),Kobayashi, Y. et al. (European Patent 335597); Weiner, A. L. et al. (PCTApplication WO89/05151); Hope, M. J. et al. (PCT ApplicationWO87/07530), all herein incorporated by reference.

In a second embodiment, liposome formulations and methods that permitintracellular uptake of the SDI-1 molecule will be employed. Suitablemethods are known in the art, see, for example, Chicz, R. M. et al. (PCTApplication WO 94/04557), Jaysena, S. D. et al. (PCT ApplicationWO93/12234), Yarosh, D. B. (U.S. Pat. No. 5,190,762), Callahan, M. V. etal. (U.S. Pat. No. 30 5,270,052) and Gonzalezro, R. J. (PCT Application91/05771), all herein incorporated by reference.

The SDI pharmaceutical compositions used for therapeutic administrationmay be sterilized, as by filtration through sterile filtration membranes(e.g., 0.2 micron membranes). The compositions may be stored inlyophilized form or as a liquid solution. It will be understood that useof certain of the foregoing excipients, carriers, or stabilizers willresult in the formation of salts of the SDI molecules.

The compositions of the present invention can be applied topically as tothe skin, or to gastrointestinal, vaginal, oral, etc. mucosa. Whenapplied topically, the SDI molecule(s) of the composition may besuitably combined with other ingredients, such as carriers and/oradjuvants. There are no limitations on the nature of such otheringredients, except that they must be pharmaceutically acceptable andefficacious for their intended administration, and cannot degrade theactivity of the active ingredients of the composition. Examples ofsuitable vehicles include ointments, creams, gels, or suspensions, withor without purified collagen. The compositions also may be impregnatedinto transdermal patches, plasters, and bandages, preferably in liquidor semi-liquid form.

For obtaining a gel formulation, the SDI molecule(s) of the compositionformulated in a liquid composition may be mixed with an effective amountof a water-soluble polysaccharide or synthetic polymer such aspolyethylene glycol to form a gel of the proper viscosity to be appliedtopically. The polysaccharide that may be used includes, for example,cellulose derivatives such as etherified cellulose derivatives,including alkyl celluloses, hydroxyalkyl celluloses, andalkylhydroxyalkyl celluloses, for example, methylcellulose, hydroxyethylcellulose, carboxymethyl cellulose, hydroxypropyl methylcellulose, andhydroxypropyl cellulose; starch and fractionated starch; agar; alginicacid and alginates; gum arabic; pullullan; agarose; carrageenan;dextrans; dextrins; fructans; inulin; mannans; xylans; arabinans;chitosans; glycogens; glucans; and synthetic biopolymers; as well asgums such as xanthan gum; guar gum; locust bean gum; gum arabic;tragacanth gum; and karaya gum; and derivatives and mixtures thereof.The preferred gelling agent herein is one that is inert to biologicalsystems, nontoxic, simple to prepare, and not too runny or viscous, andwill not destabilize the SDI molecule(s) held within it. Preferably thepolysaccharide is an etherified cellulose derivative, more preferablyone that is well defined, purified, and listed in USP, e.g.,methylcellulose and the hydroxyalkyl cellulose derivatives, such ashydroxypropyl cellulose, hydroxyethyl cellulose, and hydroxypropylmethylcellulose. Most preferred herein is methylcellulose.

The polyethylene glycol useful for gelling is typically a mixture of lowand high molecular weight polyethylene glycols to obtain the properviscosity. For example, a mixture of a polyethylene glycol of molecularweight 400-600 with one of molecular weight 1500 would be effective forthis purpose when mixed in the proper ratio to obtain a paste.

The compositions of the present invention can also be formulated foradministration parenterally by injection, rapid infusion, nasopharyngealabsorption (intranasopharangeally), dermoabsorption, or orally. Thecompositions may alternatively be administered intramuscularly, orintravenously. Compositions for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Carriers, adjuncts or occlusive dressings can beused to increase tissue permeability and enhance antigen absorption.Liquid dosage forms for oral administration may generally comprise aliposome solution containing the liquid dosage form. Suitable forms forsuspending liposomes include emulsions, suspensions, solutions, syrups,and elixirs containing inert diluents commonly used in the art, such aspurified water. Besides the inert diluents, such compositions can alsoinclude wetting agents, emulsifying and suspending agents, orsweetening, flavoring, coloring or perfuming agents.

If methylcellulose is employed in the gel, preferably it comprises about2-5%, more preferably about 3%, of the gel and the SDI molecule(s) ofthe composition is present in an amount of about 300-1000 μg per ml ofgel. The dosage to be employed is dependent upon the factors describedabove. As a general proposition, the SDI molecule(s) of the compositionis formulated and delivered to the target site or tissue at a dosagecapable of establishing in the tissue a maximum dose that is efficaciousbut not unduly toxic.

Generally, the dosage needed to provide an effective amount of thecomposition will vary depending upon such factors as the recipient'sage, condition, sex, and extent of disease, if any, and other variableswhich can be adjusted by one of ordinary skill in the art.

Effective amounts of the compositions of the invention can vary from0.01-1,000 mg/ml per dose or application, although lesser or greateramounts can be used.

When SDI-1 nucleic acid molecules are employed (as in antisense ortriplex repression), methods of “gene therapy” are employed. Theprinciples of gene therapy are disclosed by Oldham, R. K. (In:Principles of Biotherapy, Raven Press, N.Y., 1987), and similar texts.Disclosures of the methods and uses for gene therapy are provided byBoggs, S. S. (Int. J. Cell Clon. 8:80-96 (1990)); Karson, E. M. (Biol.Reprod. 42:39-49 (1990)); Ledley, F. D., In: Biotechnology, AComprehensive Treatise, volume 7B, Gene Technology, VCH Publishers, Inc.NY, pp 399-458 (1989)); all of which references are incorporated hereinby reference. Such gene therapy can be provided to a recipient in orderto treat (i.e. suppress, or attenuate) an existing condition, or toprovide a prophylactic gene therapy to individuals who, due to inheritedgenetic mutations, or somatic cell mutation, carry a predisposition toglaucoma.

Most preferably, viral or retroviral vectors are employed for thispurpose. Examples of suitable vectors are discussed by Fletcher, F. A.et al. (J. Exper. Med. 174:837-845 (1991)), Mäkelä, T. P. et al. (Gene118:293-294 (1992)), Porgador, A. et al. (Canc. Res. 52:3679-3686(1992)), Yoshimura, K. et al. (Nucl. Acids Res. 20:3233-3240 (1992)),Lim, B. et al. (Proc. Natl. Acad. Sci. (U.S.A.) 86:8892-8896 (1989)),Ohi, S. et al. (Gene 89:279-282 1990)), and Russel, S. J. et al. (J.Virol. 66:2821-2828 (1992)).

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE 1 Creation of the cDNA Library

A cDNA library was obtained using RNA from normal human neonatalforeskin fibroblasts, such as the cell line HCA2. To do this, the cellswere grown in minimal essential medium with either Earle's or Hanks'balanced salt solution supplemented with 10% fetal bovine serum (GIBCOor Hyclone). Cells were cultured, and their in vitro life span wasdetermined, under the conditions disclosed by Smith, J. R., andBraunschweiger, K. I., J. Cell Physiol. 98:597-601 (1979), herebyincorporated by reference. Quiescent cells were made by replacing thenormal culture medium with culture medium containing 0.5% serum beforethe cells become confluent. The cells were maintained in low serumculture for up to 3 weeks.

Total cellular RNA was isolated either by the guanidiniumthiocyanate/CsCI method (Garger, S. J. et al., Biochem. Biophys. Res.Commun. 117:835-842 (1983)) or a guanidinium thiocyanate/phenol method(Chomczynski, P., and Sacchi, N., Anal. Biochem. 162:156-159 (1987),RNAzol B, Biotecx Lab. Inc. TX). Poly A+RNA was isolated by oligo (dT)cellulose column chromatography (Collaborative Res. MA).

10 μg of the poly A+RNA derived from senescent cells, as describedabove, was converted to double-stranded cDNAs by using RNase H⁻/MMLVreverse transcriptase according to the instructions of the supplier(BRL, MAD), and blunt-ended by T4 polymerase treatment. Thedouble-stranded cDNA preparations were size fractionated by agarose gelelectrophoresis, and the 2-4.5 kb fraction isolated, for insertion intoan expression vector.

The expression vector used for this purpose was a 3.4 kb plasmid,designated pcDSRαΔ (FIG. 1). Plasmid pcDSRαΔ is a derivative of theplasmid pcDSRα296, which includes the Okayama-Berg SV40 promoter and theLTR from HTLV-1 (Takebe, Y. et al., Mol. Cell. Biol. 8:466-472 (1988);provided by Dr. M. Yoshida (Cancer Inst. of Japan)). Plasmid pcDSRαΔ wasformed by removing a 336 base pair (bp) segment of the Pstl-Kpnlfragment of pcDSRα296 and replacing it with 28 bp of a Pstl-Kpnlfragment from pUC19. The resulting plasmid (pcDSRαΔ) was used as acloning and expression vector.

Plasmid pSV2cat (Gorman, C. et al., Mol. Cell. Biol. 2:1044-1051 (1982))was provided by Dr. Gretchen Darlington (Texas Children's Hospital). ThepcD vector (Okayama, H., and Berg, P., Mol. Cell. Biol. 3:280-289(1983)) was provided by Dr. H. Okayama (Osaka University, Japan); theplasmid has the chloramphenicol acetyltransferase (“CAT”) gene insertedbetween the SV40 promoter and SV40 poly A signal. pcDSRαΔ-cat wasconstructed from pcDSRαΔ by the insertion of 0.8 Kb of a HindIII-SmaIdigested SRa promoter fragment into HindIII digested pSVOcat via a twostep ligation. A very strong promoter was desired in order to allow forefficient expression screening of the cDNA library. From an analysis ofseveral mammalian expression vectors (pSV2cat, pcD-cat and pcDSRαΔ-cat,transfected into young cells), the SRa promoter was found to drive theexpression of the CAT gene at high efficiency in young cycling cells.The relative CAT activities of these plasmids were calculated bynormalizing to the amount of protein used for each reaction. Thetranscriptional efficiency was about 20-fold greater than that of theconventional pSV2 promoter, which utilizes the SV40 early gene promoter.

pCMVβ carries the E. coli β-galactosidase gene driven by the humancytomegalovirus immediate early gene promoter (MacGregor, G. R., andCaskey, C. T., Nucleic Acids Res. 17:2365 (1989); provided by Dr. GrantMacGregor, Baylor College of Medicine, TX). Plasmid pβ440, which carries443 bp of the human β-actin sequence (Nakajima-Iijima, S. et al., Proc.Natl. Acad. Sci. 82:6133-6137 (1985); provided by Dr. Kozo Makino, OsakaUniversity, Japan). Plasmid pHcGAP (Tso, J. Y. et al., Nucleic AcidsRes. 13:2485-2502 (1985)), which carries a full length humanglyceraldehyde 3 phosphate dehydrogenase (GAPDH) cDNA, was obtained fromthe American Type Culture Collection, Rockville, Md.

For cDNA antisense expression, full length cDNA fragments were excisedby BamHI digestion from the originally cloned pcDSRαΔ vector, andre-ligated in the reverse direction.

cDNAs recovered from the agarose gel were directly inserted into a calfintestine alkaline phosphatase treated SmaI site of pcDSRαΔ, andtransformed into E. coli MC1061 or DH-1. Ampicillin resistant colonieswere picked randomly and plasmid sizes determined. These procedures wererepeated until 2-4.5 kb cDNA insertions were achieved in more than 90percent of the plasmids tested. Then each E. coli colony was picked withtoothpicks and 5 colonies combined into one cDNA pool. More than 400cDNA pools were prepared, grown in 96 well microtiter plates and storedin 14% glycerol at −70° C. For DNA isolation, E. coli from each cDNApool was cultured in 200 ml, and treated by the standard methods ofethydium bromide/CsCI ultracentrifugation (Garger, S. J. et al.,Biochem. Biophys. Res. Commun. 117:835-842 (1983)) one or two times,followed by dialysis against TE (10 mM Tris pH 8.0, 1 mM EDTA) solution.

EXAMPLE 2 DEAE-dextran Mediated Transfection and Transient ExpressionScreening

Young, cycling fibroblast cells were seeded at a density of 0.9-1.2×10⁵per well in 6 well tissue culture plates or 35 mm tissue culture dishes18 h prior to transfection. Transfection was done as described byCullen, B. R., In: Guide to Molecular Cloning Techniques. Methods inEnzymology., S. L. Berger and A. R. Kimmel (ed.) Academic Press, pp.684-704 (1987); herein incorporated by reference with minormodifications as described below.

For each transfection, 100 ng of pCMVβ and 400 ng of a cDNA pool weremixed and suspended in 190 μl of phosphate buffered saline (PBS)solution and 10 μl of 10 mg/ml of DEAE-dextran (Pharmacia, MW ˜500,000)was added. 400 ng of the cloning vector plasmid, pcDSRαΔ, was used withpCMVβ as a control. After washing the cells with PBS once, DNA solutionswere added and the cells incubated for up to 45 min at 37° C. in a CO₂incubator. Then 2 ml of cell culture medium with serum, containing 64 μMchloroquine (Sigma, MO) was added directly and incubated for another 2.5h. After the chloroquine treatment, the transfection mixture was removedand the cells treated with 10% dimethyl sulfoxide in cell culture mediumwith serum for 2 min. Cells were then returned to fresh cell culturemedium with serum and incubated to allow for expression of thetransfected DNA.

18 h after transfection, 0.5 μgCi/ml of ³H-thymidine was added and theincubation continued for another 48 h. Cells were fixed by adding 25 μlof 25% of glutaraldehyde solution to the culture medium and incubatedfor 5 min at room temperature, followed by three washings with PBS.Immediately after washing, cells were treated with the X-gal reactionmixture (1 mM MgCl₂, 3 mM K₄[Fe(CN)₆], 3 mM K₃[Fe(CN)₆], 0.1% tritonX-100, and 1 mM X-gal dissolved in 0.1 M sodium phosphate buffer (pH7.5) containing 10 mM KCI) for up to 20 min to allow light-blue stainingof the cells. After the X-gal staining, the cells were washed withwater, dried and processed for autoradiography using Kodak NTB nucleartrack emulsion (Kodak, NY). DNA synthesis activity in X-gal positivecells was then determined. The percent inhibition of DNA synthesis wascalculated using the formula: $\frac{\begin{bmatrix}{\% \quad {labeled}\quad {nuclei}\quad {in}} \\{\quad {{blue}\quad {cells}\quad {in}\quad {which}}} \\{\quad {{control}\quad {{plasmid}s}}} \\{\quad {{were}\quad {transfected}}}\end{bmatrix} - \begin{bmatrix}{\% \quad {labeled}\quad {nuclei}\quad {in}} \\{\quad {{blue}\quad {cells}\quad {in}\quad {which}}} \\{\quad {{cDNA}\quad {plasmids}}} \\{\quad {{were}\quad {transfected}}}\end{bmatrix}}{\begin{bmatrix}{\% \quad {labeled}\quad {nuclei}\quad {in}\quad {blue}\quad {cells}\quad {in}\quad {which}} \\{\quad {{control}\quad {plasmids}\quad {were}\quad {transfected}}}\end{bmatrix}} \times 100$

Candidate cDNA pools were divided into individual cDNAs and screenedfurther for the identification of specific DNA synthesis inhibitory cDNAsequences.

Nuclear microinjection of young cycling cells was performed as describedby (Lumpkin, C. K. et al., Mol. Cell Biol. 6:2990-2993 (1986), hereinincorporated by reference). Briefly, 5,000-10,000 cells were plated onto22 mm square etched grid coverslips (Bellco) in 35 mm tissue culturedishes. Three or four days later, nuclear microinjections were performedon a minimum of 300 cells, using either pCMVβ+cDNA plasmid orpCMVβ+pcDSRαΔ (which served as the control). Plasmids wereco-microinjected at a concentration of 50 ng/μl each. 18 hours aftermicroinjection, the cells were labeled with ³H-thymidine for 24 h,fixed, stained with X-gal and processed for autoradiography. The percentinhibition of DNA synthesis was calculated as above.

Northern blot analysis was performed using either 5 μg of total RNA or 1μg poly A+RNA. The RNA was size fractionated by electrophoresis onformaldehyde-agarose gels and transferred to nylon membranes (ICN;Biotrans, formerly Pall Biodyne A) as described by Maniatis, T. et al.,Molecular cloning: A Laboratory Manual; Cold Spring Harbor Laboratory,Cold Spring Harbor, N.Y. (1982), herein incorporated by reference.Radioactive probes were prepared by the random primer method, and blotshybridized as described by Maniatis, T. et al., Molecular cloning: ALaboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1982).

The northern blot analyses revealed that the sizes of the cellulartranscripts of the SDIs were compatible with the sizes of the SDI cDNAs.This was expected since successful expression screening requiresfull-length cDNA insertions into the vector.

For rehybridization with β-actin or glyceraldehyde phosphatedehydrogenase (GAPDH) probe, filters were repeatedly stripped of thelabelled probes following the manufacturer's instructions. The data werequantitated by an Ambis Radioanalytic Scanning System.

An assay of CAT activity was determined as follows: Young cycling cellswere seeded into 35 mm dishes and 500 ng of plasmid transfected asdescribed above. 24 hours after the transfection, the cells were scrapedfrom the dish, and CAT assay 35 performed as described by Gorman(Gorman, C., In: DNA Cloning, A Practical Approach. IRL Press, Oxford,England, pp. 143-164 (1985), herein incorporated by reference).

EXAMPLE 3 cDNA Cloning of the Senescent Cell Derived Inhibitors (SDI) ofDNA Synthesis

Double-stranded cDNAs were synthesized from senescent cell derived polyA+RNA, which has been shown to inhibit DNA synthesis in young cells whenmicroinjected into the cytoplasm (Lumpkin, C. K. et al., Science232:393-395 (1986)). The cDNAs were size fractionated, inserted intopcDSRαΔ The resulting E. coli clones were divided into small pools.Plasmids from each pool were co-transfected with the transfection markerplasmid, pCMVβ, which allowed a determination of the labelling index oftransfected cells specifically, since even in high efficiencytransfection, frequencies varied from experiment to experiment.Transfection frequencies of the marker plasmid ranged from 30-90%. About200 cDNA pools were screened and four pools remained positive for DNAsynthesis inhibitory activity after five repeated transfections. Thecandidate pools were then divided into individual plasmids and screenedfurther.

Three independent positive plasmid clones were obtained. In the cDNApool A, only one plasmid, No. 2, exhibited strong DNA synthesisinhibitory activity. Similarly, in pools B and C only one cDNA clonecaused inhibition. The size of inserted cDNAs was 2.1 kb, 1.2 kb and 2.7kb, respectively. These cDNA sequences have been designated as senescentcell derived inhibitors, SDI-1, SDI-2 and SDI-3, respectively.

The nucleotide sequence of the SDI-1 cDNA clone (SEQ ID NO: 1), and theamino acid sequence of SDI-1 (SEQ ID NO: 2) have been determined. ThecDNA sequence presented herein for SDI-1 differs from that described inU.S. patent application Ser. No. 07/808,523 in possessing an unrecited Gat position 286, and in having the sequence CG rather than GC atposition 1843-1844. The presently disclosed sequence was obtainedthrough the re-sequencing of the pcDSRαΔ-SDI-1 plasmid whose isolationand characteristics were described in U.S. patent application Ser. No.07/808,523. E. coli DH5 transformed with the pcDSRαΔ-SDI-1 plasmid wasdeposited with the American Type Culture Collection, Rockville, Md.,USA, on Oct. 1, 1992, and has been accorded accession number ATCC 69081.

A nucleic acid molecule whose sequence corresonds to a portion of theSDI-1 nucleotide sequence reported herein has been identified among the2375 random gene sequence fragments reported by Adams, M. D. et al.(Nature 355:632-634 (1992)).

EXAMPLE 4 Microinjection of SDI Sequences into Young Cycling Cells

In order to verify the functional activity of SDI sequences,microinjections were performed. A plasmid carrying either SDI-1 or SDI-2was co-microinjected with the marker plasmid into the nuclei of youngcycling cells. The labelling index of the resulting blue cells wasdetermined (Table 1). These plasmids showed strong inhibitory activityon DNA synthesis of young cells. For control experiments, the emptyvector was co-microinjected with the marker plasmid. This caused slightinhibition when the labelling index was compared with uninjected cells,a phenomenon also observed in transfection experiments. Microinjectionswith SDI-3 were not performed because the inhibitory activity was lowerthan SD-I and SD-2 transfection experiments.

In addition to normal human fibroblasts, the SDI-1 molecules were alsofound to be capable of inhibiting the synthesis of DNA in several tumorcell types (melanoma, lung carcinoma, and ovarian tumor), and inimmortalized SV40-transformed fibroblasts, and CHO cells. SDI-1molecules were also capable of inhibiting the synthesis of DNA in normalbovine pulmonary artery smooth muscle.

TABLE 1 No. of Labelled No. of Nuclei per Labelling Plasmids Cells TotalBlue Index % Injected Injected Cells* (%) Inhibition Exp. pCMVβ+ 33558/97 59.8 0 1 pcDSRαΔ† pCMVβ+ 380 20/89 22.5 62.4 SDI-1 pCMVβ+ 380 6/82 7.3 87.8 SDI-2 Exp. pCMVβ 423  68/109 62.3 0 2 +pcDSRαΔ† pCMVβ+465 26/98 26.5 57.5 SPI-1 pCMVβ+ 475  27/118 22.9 63.2 SDI-2 Notes: †Control; *The number of cells expressing detectable levels ofβ-galactosidase; The concentration of each DNA was 50 μg/ml.

EXAMPLE 5 Antisense DNA Transfection

In order to examine whether any inhibitory activities are sequenceorientation specific, antisense expression vectors of SDI-1 and SDI-2sequences were constructed. Since both sequences lacked BamHI sites andsince BamHI sites were present at both ends of the cDNA (FIG. 1), thesequences were easily excised and religated in the opposite orientation.Transfection of antisense sequences resulted in no inhibition of DNAsynthesis in young cells (FIG. 3). In addition, no enhancement wasobserved. The results clearly indicate the sequence orientationspecificity of the SDI activity, and suggest the presence of specificgene products coded by the cDNA sequences.

Normal human fibroblasts and many other cells cease to synthesize DNA inthe absence of appropriate growth factors. Since SDI-1 is a key negativeregulator of initiation of DNA synthesis, molecules that are antisenseto SDI-1 are able to cause cells to enter S phase. To demonstrate thisability, several normal human cell lines were isolated and provided withSDI-1 antisense molecules (expressed by cloning the SDI-1 cDNA in andantisense orientation into a vector having a metallothionein promoter).

For this purpose, antisense expression vectors were constructed bycloning SDI-1 antisense sequences into PMET, an inducible expressionvector containing an altered human metallothionine promoter. Themetallothionine promoter in PMET was derived from pM26 and contains adeletion in the basal promoter and the addition of synthetic metalresponse elements in triplicate (McNeall, J. et al., Gene 76:81-88(1989)). For construction of PMET, adenovirus sequences containing E1Agene 12S and 13S introns (nucleotides 917-1673) were first cloned intothe mammalian expression vector PRC/CMV (Invitrogen) at the Not I/Apa Isites of the multiple cloning region of this plasmid to createpRc/CMV-Ad. To ensure that no translation of E1A sequences occurred andto create an Spe I cloning site in this vector, an SpeI linkercontaining a stop codon was inserted between the CMV promoter and E1Asplice sequence in-frame with the E1A sequence. The CMV promoter ofplasmid Rc/CMV-Ad was then replaced with the pM26 promoter to createPMET. To accomplish this, the metallothionine promoter was excised frompM26 by BgIII digestion and inserted into the BamHI site of pBlueScript(pBS; Stratagene). An EcoRV and NotI fragment containing the promoterwas then subcloned from pBS into a filled-in BgIII site and NotI site ofRc/CMV-Ad. Antisense sequences from the SDI-1 CDNA were derived from thefull-length CDNA cloned into the BamHI site of pBS. The StuI site atnucleotide 127 was converted to an SpeI site by insertion of an SpeIlinker to make SDISPE127. An SpeI linker was also inserted at the ApaIsite at nucleotide 318 to create SDISPE318. These constructs weredigested with SpeI and inserted in antisense orientation into the SpeIcloning site of PMET, thus creating pMET-AS127 and pMET-AS318.

Cell lines expressing SDI-1 antisense were obtained by calcium phosphatetransfection using the BES/CaPO₄ procedure described by Chen, C. et al.,Molec. Cell. Biol. 7:2745-2752 (1987)) except that the cells were notreplated after transfection. HCA2 cells (3×10⁵) at PD10 were transfectedwith 20 μg PMET, pMETAS127 or pMETAS318 DNA. Following two weeks of G418selection, colonies were picked and expanded. Inducibilty and integrityof the inserted sequence was examined by the addition of 100 μM ZnCl₂and 2 μM CdCl₂ followed by RNA analysis. RNA analysis of total cell RNAfrom stable transformants was performed by RNAse protection usingantisense RNA probes internally labeled with [³²P]-UTP as described byAdami, G. et al., EMBO J. 10:3457-3465 (1991)). The probe pMET+SDI-1,containing SDI-1 nucleotides 444-686, was used to measure expression ofSDI-1 from both the transfected genes and endogenous mRNA. After EcoNIdigestion, transcription from the SP6 promoter in the PMET vectorresults in a antisense labelled probe that hybridizes to both RNA fromthe expression expression vector and to endogenous SDI-1 mRNA. Thisallows a comparison of relative levels of expression from the introducedconstruct and SDI-1 endogenous RNA. As a control β-actin mRNA wasmeasured in all assays. The actin probe contains nuclotides 2124-2189inserted between the Eco Ri and the filled-in Bam Hi sites of pBS.

When the stably transfected cells were placed in medium containinglowered amounts of growth factors (0.5% fetal bovine serum) for 7-10days and pulse labelled for 24 hours with tritiated thymidine, fewerthan 5% incorporated label. However, when antisense SDI-1 was induced bythe addition of ZnCl₂ and CdCl₂ 24 hours prior to the addition of thetritiated thymidine, more than 25% of the cells were found, byincorporation of label, to have initiated the synthesis of new DNA, andthus to have regained the capacity to proliferate.

This experiment shows that the antisense sequences of the presentinvention can be used to immortalize cells that would absent suchtreatment undergo sensecnece.

EXAMPLE 6 Expression of SDI mRNAS During Cellular Senescence

To examine the changes in SDI mRNA expression during cellularsenescence, total RNA from young and senescent cells was hybridized to32P-labelled SDI cDNA probes. The SDI-1 probe hybridized to a 2.1 kbcellular transcript, SDI-2 hybridized to a 1.4 kb transcript, and SDI-3hybridized to a 2.5 kb transcript (Table 2). Table 2 provides aquantitation of the total RNA northern analysis of expression of SDIgenes in young (Y) and senescent (S) cells. 5 μg each of total RNA fromyoung and senescent cells were hybridized with SDI probes. The filterswere repeatedly stripped of the radioactive probe and rehybridized withthe probes for the internal controls. The relative amount of SDI mRNA ineach sample was normalized by the amount of GAPDH detected on the samefilter and by the relative amount of SDI/GAPDH.

TABLE 2 Quantitation of the Northern Analysis SDI-1 SDI-2 SDI-3ATTRIBUTE Y S Y S Y S Relative Amount of 1.0 3.3 1.0 0.31 1.0 0.31 SDIRelative Amount of 1.0 0.37 1.0 0.36 1.0 0.38 GAPDH Relative Amount of1.0 9.3 1.0 0.86 1.0 0.82 SDI/GAPDH

During cellular senescence, the SDI-1 message increased about 3-fold,while SDI-2 and SDI-3 messages decreased 3-fold. The same filters wererehybridized with a β-actin, and then to a GAPDH probe as internalcontrols. The results demonstrated that expression of both control genesdecreased about 3-fold during cellular senescence. In previous studies,a 2-3 fold decrease of β-actin expression during cellular senescence hadbeen observed (Kumazaki, T. et al., Exp. Cell Res. 195:13-19 (1991);Seshadri, T., and Campisi, J., Science 247:205-209 (1990); Furth, J. J.,J. Gerontol. 46:B122-124 (1991)). The decreased expression of bothβ-actin and GAPDH genes in senescent cells led to the use of poly A+RNAfor northern analysis. Poly A+RNA was isolated from the total cellularRNA preparations used for Table 2, and hybridized to SDI cDNA, followedby probing with β-actin and GAPDH respectively (Table 3). Table 3discloses the results of a poly A+RNA Northern analysis of SDI geneexpression in young (Y) and senescent (S) cells. 1 μg each of poly A+RNAfrom young and senescent cells were used for the analyses. The relativeamount of SDI mRNA in each sample was calculated as in Table 2.

TABLE 3 Quantitation of the Northern Analysis SDI-1 SDI-2 SDI-3ATTRIBUTE Y S Y S Y S Relative Amount 1.0 0.83 1.0 0.87 1.0 0.87 ofGAPDH Relative Amount 1.0 11.4 1.0 1.0 1.0 1.0 of SDI/GAPDH

The results clearly indicated that the expression of both β-actin andGAPDH was equal in young and senescent cells when they were compared onthe basis of mRNA, consistent with previous observations. When SDI geneexpression was compared at the mRNA level, SDI-1 mRNA was increased11-fold in senescent cells, whereas expression of SDI-2 and SDI-3remained constant throughout the in vitro lifespan (Table 3). Thisresult suggests that SDI-1 is a senescent cell specific inhibitor of DNAsynthesis, whereas SDI-2 and SDI-3 are most likely more generalinhibitors involved in cell cycle regulation.

EXAMPLE 7 Changes of Poly a RNA Content During Cellular Senescence

The observation that the results of the total versus poly A+RNA northernanalyses were quantitatively different, indicated that the poly A+RNAcontent in total RNA preparations might change during cellularsenescence. To test this hypothesis, cells were cultivated serially andtotal RNA was harvested at different population doubling levels. PolyA+RNA was isolated from each sample.

The result clearly indicated that poly A+RNA content decreased graduallyduring cellular senescence (FIG. 4). In FIG. 4, cells were cultivatedserially and total RNA was harvested. Poly A+RNA: % of total RNA wasplotted against the culture's age (% in vitro life span completed).Senescent cells had 3-4 fold less poly A+RNA when compared with veryyoung cells. However, when total RNA content per cell was calculated,senescent cells had 1.3-1.5 fold more than young cells (see, Cristofalo,V. J., and Kritchevsky, D., Med. Exp. 19:313-320 (1969)).

In order to determine whether SDI-1 message increased gradually duringsubcultivation or whether a rapid increase occurred near the end of thein vitro life span, poly A+RNA from cultures at different populationdoublings was hybridized with the 32_(p) labelled SDI-1 probe. Thisanalysis revealed that SDI-1 expression increased as the cultures becamesenescent, with a major change occurring during the final few passages(Table 4). Table 4 shows the accumulation of SDI-I mRNA during cellularaging process. One microgram each of poly A+RNA from the cells ofdifferent population doublings were hybridized to SDI-1 probe. Therelative amount of SDI-1 mRNA in each sample was calculated as in Table2.

TABLE 4 Quantitation of % Lifespan Completed ATTRIBUTE 24% 37% 46% 66%78% 88% 100% Relative Amount 1.0 1.6 1.5 1.3 1.4 1.3 0.9 of GAPDHRelative Amount 1.0 2.2 2.1 4.0 3.5 6.2 20.5 of SDI/GAPDH

Changes in SDI-1 expression during quiescence were also examined. Young,quiescent cells were maintained in 0.5% fetal bovine serum(FBS)-containing medium for up to three weeks. Total RNA was harvestedeach week and the amount of RNA hybridizing to the SDI-1 probe wasanalyzed. SDI-1 message increased significantly during cellularquiescence (Table 5). Table 5 shows the accumulation of SDI-1 mRNAduring cellular quiescence. 4 μg each of total RNA was obtained from theyoung cells cultured with 0.5% FBS containing medium for 1, 2, 3 weeks,was hybridized with SDI-1 probe. The relative amount of SDI-1 mRNA wascalculated as in Table 2 (C: control culture with 10% FBS medium). Whenthe result was normalized to GAPDH expression, SDI-1 expression wasfound to have increased 18-fold after two weeks in low serum mediumcompared to that of a control dividing culture in 10% FBS medium.

TABLE 5 Accumulation of SDI-1 mRNA During Cellular Quiescence ATTRIBUTEC 1 wk 2 wk 3 wk Relative Amount of 1.0 0.72 0.88 0.37 GAPDH RelativeAmount of 1.0 12.2 18.4 14.9 SDI/GAPDH

The fact that the cellular representation of mRNA vs total RNA was foundto change during cellular senescence is significant. During the in vitroaging process, the content of mRNA was found to decrease gradually (FIG.4), in spite of the slight increase of the total RNA per cell. Thisphenomenon indicates that a gradual decline of the overall geneexpressions during the cellular aging process, and explains thedecreased expression of β-actin and GAPDH genes in senescent cells whenNorthern blot analysis was done with total RNA (Table 2). However, theexpression levels of these housekeeping genes between young andsenescent cells were almost constant when the Northern blot analysis wasdone with poly A+RNAs (Table 3). This analysis revealed the strongexpression of SDI-1 message in senescent cells, and unchangingexpression of SDI-2 and 3 genes throughout the in vitro life span.

EXAMPLE 8 The SDI-b 1 Gene

The SDI-1 gene codes for a senescent cell specific inhibitor of DNAsynthesis. Increased expression of this gene occurred when the cellsentered their final few divisions (Table 4). The expression kineticscorrelated well with the phenotypic expression of senescence cells.SDI-1 gene expression was also found to increase after young cells weremade quiescent and nondividing by serum deprivation (Table 5). Thisresult demonstrates the involvement of this gene in the inhibition ofDNA synthesis of cellular quiescence as well as senescence. Cells madequiescent by deprivation of serum growth factors have been shown toproduce an inhibitor of DNA synthesis with characteristics similar tothe inhibitor from senescent cells (Pereira-Smith, O. M. et al., Exp.Cell Res. 160:297-306 (1985); Stein, G. H., and Atkins, L., Proc. Natl.Acad. Sci. USA. 83:9030-9034 (1 986)).

The fact that SDI-1 expression increases during both senescence andquiescence indicates that it is an inhibitor of DNA synthesis (Smith, J.R., J. Gerontol. 45:B32-35 (1990); herein incorporated by reference).Alternatively, SDI-1 sequences might be related to the growtharrest-specific genes recently cloned from mouse cells (Schneider, C. etal., Cell 54:787-793 (1988); Manfioletti, G. et al., Mol. Cell. Biol.10:2924-2930 (1990)).

EXAMPLE 9 The Expression of the SDI-1 Gene Product

SDI-1 CDNA has been expressed in two different bacterial expressionsystems, has been transcribed in vitro and translated in two differentin vitro systems. Two bacterial expression systems were used in order tomaximize the probability of obtaining sufficient amounts of SDI-1protein. In the first expression system, SDI-1 protein was expressed asa glutathione S-transferase fusion protein at yields of 5-10 μg perliter of bacterial culture. The recombinant protein could be cleavedwith thrombin and purified in order to give an SDI-1 protein with a fewextra amino acids. The GST fusion was formed by cleaving a Schistosomajaponicum GST-encoding polynucleotide with BamHI so as to produce acleavage fragment that contained nucleotides 1-673 of the GST-codingsequence. The free BamHI site at position 673 generated via suchtreatment was then ligated to the SDI-1 encoding polynucleotide in orderto form the GST-SDI-1 gene fusion. The GST-SDI-1 fusion protein wasproduced via recombinant expression of this gene fusion.

In the second expression system, a 6 histidine amino terminal tag wasutilized in order to aid in purification. This recombinant protein maybe used without further modification. Both systems permitted theisolation of pure preparations of protein.

In the course of this experiment, in vitro transcription and translationsystems were used to confirm the open reading frame deduced from thenucleic acid sequence of the SDI-1 cDNA. The calculated molecular weightof the SDI-1 protein is approximately 16,000 daltons. The in vitrosynthesized protein migrates, by SDS PAGE, with a relative mobility ofapproximately 21,000 daltons. This small difference may be due to aslightly unusual charge or conformation of the SDI-1 protein. A partialamino acid sequence of the bacterially expressed protein verified theopen reading frame (SEQ ID NO:2).

The bacterially expressed proteins were used to generate polyclonalantisera and monoclonal antibodies to the intact native protein. Suchantibodies may be more effective in immunoprecipitation of SDI-1 proteinand SDI-1 protein complexes than the antisera produced from syntheticpeptides. Preliminary immunocytochemical studies, using an antisera ofhighest affinity (antisera #55) which reacted strongly with the fusionprotein on a western transfer at a 1:20,000 dilution, suggested that theSDI-1 protein was relatively abundant in senescent cells compared todividing young cells. In senescent cells the location appears to beperinuclear, whereas in young cells there appears to be a small amountof SDI-1 protein located in the nucleus. In order to obtain specificstaining it was necessary to pre-absorb the antisera against a fixedcell monolayer of cells which do not express detectable levels of SDI-1mRNA (TE85). The cells were fixed with 4% paraformaldehyde followed bymethanol.

In order to study the cellular phenotype resulting from the inducedexpression of SDI-1 mRNA in cells which normally express the gene at lowlevels and to examine the effect of antisense SDI-1 constructs it isdesirable to obtain cell lines in which the SDI-1 gene is stablyintegrated under the control of an inducible promoter. Toward this goal,a functional vector containing SDI-1 under the control of themetallothionine promoter was constructed. Following transfection of thisconstruct into young proliferation competent cells and incubation in thepresence of 100 μM zinc chloride and 2 μM cadmium chloride, initiationof DNA synthesis was inhibited by about 50%. In the absence of metalsthere was no inhibition of DNA synthesis. The inhibitory activityobserved is not due to metal toxicity since cells transfected with thecontrol vector (pcDSRα) and grown in the presence of metals were foundto have approximately 90% of the DNA synthetic capacity of cellstransfected with the same plasmid grown in the absence of metals.

In order to demonstrate that the inhibitory effects observed with SDI-1were not related to the nature of the specific promoter used to driveexpression, the capacity of SDI-1, expressed from other promoters, toinhibit DNA synthesis was investigated. Young proliferating humanfibroblasts were therefore co-transfected with CMV-β-gal and CMV-SDI-1.Transfection of cells with CMV-β-gal had little effect on DNA synthesiswhile CMV-SDI-1 was even more effective than SDI-1 in the pcDSRα vectorin these particular experiments.

The SV40 large T antigen is capable of inducing senescent cells tosynthesize DNA. It was therefore of interest to determine whether theinhibitory action of SDI-1 could be overcome by the expression of Tantigen. Moreover, it was desirable to determine that the action ofSDI-1 was not due to the induction of a general metabolic imbalance incells. If such were the case, one would not expect large T antigen toantagonize its effect. For these reasons, cells were co-transfected withSDI-1 cDNA and vectors in which T antigen was driven by the CMVpromoter. Such co-transfection experiments revealed that the inhibitoryactivity of SDI-1 was largely abolished by the co- expression of theSV40 large T antigen.

Transient transfection assays were performed using an additional normalhuman fibroblast cell line (neonatal foreskin cell line (CSC303) and theWI38 immortal cell line in order to determine the generality of theinhibitory effect of SDI-1. In both cases, significant inhibition(40-50%) was observed. Furthermore, SDI-1 was found to inhibit SUSMI(40%) but not an SV40 transformed cell line GM639 or HeLa cells (<20%).The results thus far are consistent with earlier results obtained fromheterokaryon experiments in which HeLa cells and cells transformed withSV40 virus were not inhibited by fusion with senescent cells. Thisprovides further evidence that SDI-1 behaves like the inhibitorpreviously detected in senescent cells.

EXAMPLE 10 Southern Analysis of the SDI-1 Gene

In order to determine whether the absence or inactivity of SDI-1 wasresponsible for cellular immortality in any of the four complementationgroups for indefinite division, genomic DNA and mRNA was examined fromcell lines representative of the four groups. Southern analysis revealedthe expected 5 and 10 kb bands after digestion with EcoRI. Therefore, nogross deletions or rearrangements have occurred in the SDI-1 gene inthese cell lines. By Northern analysis, it was determined that SDI-1mRNA was lower or absent in the cell lines that had been assigned tocomplementation groups B and C. SDI-1 was present at higher levels incell lines representative of complementation groups A and D. Thisresults suggests that part of the mechanism by which the cell lines mayhave escaped cellular senescence is through the loss of ability toexpress sufficient levels of the active SDI-1 gene.

EXAMPLE 11 Characterization of SDI Sequences

Using a functional screening method, a novel DNA synthesis inhibitorygene, SDI-1, was identified. The gene is expressed at high levels innonproliferating human diploid fibroblasts. Message levels of SDI-1increased 10 to 20-fold as normal human cell cultures were aged invitro, with the expression kinetics correlating closely with thephenotypic expression of cellular senescence. In addition, SDI-1 messageincreased when cells were made quiescent by growth factor deprivation.

The results described above demonstrate that SDI-1 codes for a novel,physiologically active gene product that is important for cell cyclecontrol. Expression of the gene is modulated during exit from G₀ andentry into S phase in cells that have been stimulated to enter the cellcycle. In addition, expression of antisense SDI-1 message stimulatescells to enter the cell cycle in the absence of growth factors. Theobservation that SV40 T antigen can counteract the inhibitory activityof SDI-1 in a manner similar to that observed with the negative growthregulators p53 and Rb (Lane, D. P. et al., Nature 278:261-263 (1979);Linzer, D. I., H. et al., Cell 17:43-52 (1979); De Caprio, J. A. et al.,Cell 54:275-283 (1988)), underscores the importance of this gene productin the regulation of the cell cycle.

The recombinant SDI-1 protein of the present invention inhibits thephosphorylation of histone H1 by CDK2. Since SDI-1 blocks DNA synthesis,this finding indicates that the phosporylation of histone H1 has a rolein the initiation of DNA synthesis.

The SDI-1 gene product is also a potent inhibitor of severalcyclin-dependent kinases, including CDC2, CDK2, and CDK4. In similarexperiments using human cell extracts, recombinant SDI-1 was found toinhibit CDK2 kinase activity. These results are of particular importancein view of what is known about the various proteins involved in cellcycle progression. Several human G1 cyclin candidates (cyclins C, D, andE), identified by their ability to complement a budding yeast strainthat lacked G1 cyclins (Xiong, Y. et al., Cell 65:691-699 (1991); Lew,D. J. et al., Cell 65:1197-1206 (1991); Xiong, Y. et al., Curr. Biol.1:362-364 (1991); Koff, A. et al., Cell 66:1217-1228 (1991)), were foundto be cell cycle regulated, with maximal mRNA expression occurring atdifferent points in GI (Lew, D. J. et al., Cell 65:1197-1206 (1991)).Since D-type cyclins and cyclin E are associated with active kinasecomplexes (Koff, A. et al., Cell 66:1217-1228 (1991); 1992; Dulic, V. etal., Science 257:1958-1961 (1992); Matsushime, H. et al., Cell65:701-7139 (1991); Ewen, M. E. et al., Cell 73:487-4976 (1993); Kato,J. Y. et al., Genes Devel. 7:331-342 (1993), it is likely that thesekinases have a role in the commitment of mammalian cells to a new roundof cell division at the “restriction point.” (Pardee, A. B., Science246:603-608 (1989)). Indeed, recent reports indicate that cyclin E-CDK2kinase complexes have maximal activity in late G1 and early S phase(Dulic, V. et al., Science 257:1958-1961 (1992); Koff, A. et al., Cell66:1217-1228 (1991)), and also have the ability to phosphorylate the RBprotein in cultured human cells (Hinds, P. W. et al., Cell 70:993-1006(1992)) and in vitro (Ewen, M. E. et al., Cell 73:487-4976 (1993)). Thissuggests that the kinase may play a pivotal role in the regulation ofthe G1-to-S phase transition of the cell cycle.

Immunoblots of SDI-1 protein have revealed that levels of this proteindo not appear to vary extensively in cells in different growth states(ie. actively growing versus quiescent or senescent cells). However,consistently higher amounts of protein are present in non-dividingcompared with proliferating cells. This seems reasonable because SDI-1is a potent negative regulator of CDK activity, and tight regulation ofthis inhibitor would be essential for proper cell cycle regulation andprogression. Small changes in the amount of inhibitor protein couldresult in a major impact on the various gene products it controls. Atleast two CDKs: CDC2 and CDK2, maintain relatively constant steady-stateprotein levels through the cell cycle despite cell cycle phase-dependentchanges in mRNA. SDI-1 may be regulated in a similar manner, such thatthe level of SDI-1 protein is precisely controlled at a particularlevel, and that new CDK/cyclin synthesis and activation is needed toovercome the inhibitory effects of SDI-1 to allow for progressionthrough the cell cycle. Thus, SDI-1 would prevent entry into the cellcycle until a required threshold of stimulatory gene products werepresent, allowing the cell to proceed through the “restriction point” ofthe cell cycle. Such a dynamic equilibrium between active CDK/cyclincomplexes and the inhibitor SDI-1 protein explains the observedstimulation of DNA synthesis in quiescent cells following a smalldecrease in the steady-state levels of SDI-1 protein due to antisenseSDI-1 mRNA expression. Thus, SDI-1 may function in the cell in a mannersimilar to other cell proliferation inhibitors, such as the tumorsuppressor genes p53 and Rb, and the SDI-1 gene may be a target formutation in various tumors.

Although senescent cells cannot be stimulated to enter S phase by theaddition of mitogens, they do express mRNAs for many cellcycle-regulated genes including cyclins D1, cyclin E, CDK2, Rb, p53,c-H-ras, c-myc, c-jun, and jun B. However, several other important cellcycle-regulated genes, including c-fos, histone H3, CDC2, cyclin A,cyclin B1, and PCNA, are not expressed in mitogen-stimulated senescentcells. The lack of phosphorylation of the protein product of theretinoblastoma susceptibility gene Rb in senescent cells could be onecause for the inability of senescent cells to synthesize DNA. However,cyclin E-CDK2 complexes, though relatively abundant in senescent cells,lack the kinase activity which could potentially phosphorylate Rb invivo.

The SDI molecules of the present invention are expressed at a higherlevel in senescent than in actively cycling cells. Thus, lack of properCDK activity through the regulatory action of SDI-1 could be a keyreason for the inability of senescent cells to enter S phase. This issupported by the fact that senescent cells are primarily deficient inevents downstream of the postulated SDI-1 mediated inhibition of CDK2.

Overexpression of E2F-1, a component of the E2F-1 transcription factorwhich has a wide range of target genes, was found to be capable ofreversing the inhibitory effect of SDI-1. It is well established thatthe tumor suppressor gene Rb, as well as the related p107 proteincomplexes with E2F-1 to inhibit transcription. Overexpression of cyclinsA and E reverses pRb-mediated suppression of proliferation. In addition,overexpression of E2F-1 can induce quiescent REF-52 cells to synthesizeDNA. Thus, in view of the observation that E2F-1 reverses the negativegrowth activity of SDI-1, E2F-1 may be the last step in a cascade ofevents controlled by p53, SDI-1 and Rb.

EXAMPLE 12 the Relationship Between Cellular Tumor Suppressors and SDISequences

A role for SDI-1 in cell cycle arrest is indicated by the fact that innormal human cells made quiescent either by serum deprivation or growthto high density, SDI-1 mRNA levels were increased 10-20 fold comparedwith cycling cells. However, upon addition of serum, SDI-1 mRNA levelswere found to rapidly decrease to low levels just prior to the onset ofDNA synthesis. Thus SDI-1 appears to act as a “check point” to inhibitcell proliferation in the presence of unfavorable external conditions.Many immortal cell lines are unable to block initiation of DNA synthesisin response to insufficient growth factors. However, in accordance withthe present invention, the overexpression of SDI-1 in various immortalhuman cells resulted in inhibition of DNA synthesis in several of thecell lines regardless of their ability to arrest cell proliferation inresponse to lowered growth factors.

The physiological significance between the overexpression of SDI-1 andthe inhibition of cell proliferation by overexpression of SDI-1 isstrengthened by the finding that SDI-1 can inhibit the kinase activityof cyclin/cdk2 complexes. Indeed, as indicated the addition of 250 ng ofpurified GST-SDI-1 fusion protein to cyclin/cdk2 complexes(immunoprecipitated from HeLa cell extracts by cdk2 antisera) resultedin half maxial inhibition of histone H1 kinase activity.

In order to further investigate the molecular mechanism through whichSDI-1 mediated its anti-proliferative activity, the steady-state levelsof SDI-1 RNA were evaluated in a number of immortal cell lines,including MDAH 041. The MDAH 041 cell line was derived from aLi-Fraumeni syndrome patient, and does not synthesize p53. The cellswere found to be able to continue to synthesize DNA in the presence oflow serum growth factors.

SDI-1 levels were determined by northern analysis and were normalized toeither GAPDH or b-actin mRNA. DNA was introduced by electroporation.Rapidly growing cells were trypsinized and 10⁶ cells were resuspended in0.5 ml phosphate buffered saline along with 10 μg of carrier DNA, 1 μgof pCMVb (MacGregor, G. R. et al., Nucl. Acids Res. 17:2365 (1989))coding for the b-galactosidase gene and 1 μg pCMVSDI-1 (containingnucleotides 1-685 of SDI-1). Plasmid pCMVb served as a marker to detectcells that were capable of incorporating and expressing exogenous DNA.Following a pulse of 250-350 volts, 3×10⁵ cells were seeded into 35 mmcell culture dishes. Tritiated thymidine (1 μCi/ml) was added to theculture medium 24-30 hours after electroporation and the cells wereincubated for an additional 24 hours. The cells were fixed, stained forb-galactosidase activity, and processed for autoradiography to determinethe percentage of b-galactosidase positive cells that had synthesizedDNA in the presence of the tritiated thymidine. The percent inhibitionwas determined relative to control cells that were transfected with pCMVvector and pCMVb. Calcium transfection was used in the case of MDAH 041cells. The correlation between SDI-1 mRNA levels and DNA synthesis ispresented in Table 6. The data presented are averages of at least twoexperiments.

TABLE 6 Correlation between SDI-1 mRNA Level and Inhibition of DNAsynthesis % SDI-1 Cell Line Inhibition mRNA level P53 Status MADH 041 95± 4 Not Mutant Detectable SAOS2 47 Not Done Mutant TE85 75 ± 7 NotMutant Detectable T98G 35 ± 5 Not Mutant Detectable Hela 60 ± 5 LowHPV-18 infected A1698 15 ± 3 Normal Wild-type UABC023 57 ± 6 Low UnknownSer 31 → Arg 31 Homozygous GM639 78 Normal SV40-transformed GM847 21Normal SV40-transformed Ser31 → Arg 31 Heterozygous RN13 No InhibitionNormal Unknown PR282 No Inhibition Normal Unknown

As indicated in Table 6, SDI-1 mRNA levels were found to be very low orundetectable in several cell lines which also lacked wild type p53protein. Significantly, SDI-1 RNA and protein was undetectable the MDAH041 cells. The correlation between p53 level and SDI-1 level suggestedthat p53 mediated its tumor suppressing activity by inducing a senescentstate through the induction of SDI-1, and that cells which lacked p53were neoplastic due to their inability to induce SDI-1 expression.

In view of the well established role of p53 in cell growth control andas a transcriptional activator or suppressor, and the fact that MDAH 041cells do not express wild type p53, the finding that they also lackedSDI-1 expression indicated that the introduction of p53 into these cellswould result in an induction of SDI-1 expression and thus, in growtharrest.

To further demonstrate the ability of p53 to induce SDI-1 expression,p53 was introduced into MDAH 041 cells. Such introduction resulted inincreased expression of SDI-1 and in the inhibition of DNA synthesis, asmeasured by tritiated thymidine autoradiography. The degree ofinhibition increased with the amount of p53 plasmid introduced. If theinhibition of DNA synthesis observed was due to an induction of SDI-1 byp53, rather than by some other effect of p53, the inhibitory activitywould be lost following co-transfection of antisense SDI-1 sequences.

Therefore, to demonstrate the direct induction of SDI-1 by p53, theabove-described SDI-1 and antisense SDI-1 gene sequences wereco-transfected with a p53 gene construct into normal human fibroblasts.As expected, the antisense construct was found to eliminate 80% of theinhibition of DNA synthesis caused by SDI-1 alone. When 4 μg of SDI-1and increasing amounts of p53 plasmids were co-transfected into MDAH 041cells, the antisense SDI-1 was found to be capable of effectivelycounteracting the inhibition of DNA synthesis caused by p53 alone. Thesefindings are summarized in Table 7. This finding verified the conclusionthat one manner in which p53 causes inhibition of DNA synthesis is byactivating the expression of SDI-1 and that such induction of SDI-1 is arequisite for part of the DNA synthesis-inhibitory activity of p53. Suchactivation occurs, at least in part, by the transcriptional activationof the SDI-1 gene. The expressed SDI-1 protein acts, in part, byinhibiting the kinase activities of CDK/cyclin complexes and cantherefore act at multiple points in the cell cycle to block progression.Loss of wild type p53 activity would lead to lack of expression of SDI-1and thereby result in inappropriate cell cycle progression.

TABLE 7 The Induction of SDI-1 by P53 Amount of P53 DNA DNA Synthesis(as approx. % of control) Transfected (ng) P53 Alone P53 + anti-SDI-1 0100 121 10 25 50 30 15 45 100 10 25

Mutations in the gene encoding p53 protein are common in human tumorswith approximately 50% of tumors expressing a mutant p53. This has ledto the conclusion that p53 acts as a negative growth regulator and is atumor suppressor gene. One aspect of the present invention concerns therecognition of the molecular mechanism responsible for the anti-oncogeneactivity of p53. SDI-1 has been found to be an inhibitor of cell cycleprogression which acts at least in part by inhibiting the kinaseactivities of cdk/cyclin complexes. As such it can act at multiplepoints in the cell cycle to block progression. Since p53 is required fortranscriptional activation of SDI-1, inactivation of this function couldallow uncontrolled and inappropriate cell cycle progression. This wouldallow cells to ignore the normal external singnals for cell cycle stasisand permit proliferation in situ. Since SDI-1 is downstream of p53,SDI-1 appears to be the effector of p53 action. Furthermore, mutationshave been found in SDI-1 which may contribute to altered cellproliferation in cells without mutated p53.

EXAMPLE 13 Capacity of SDI-1 to Suppress the Proliferation of TumorCells

As indicated above, SDI-1 has the capacity to suppress the proliferationof tumor cells. To demonstrate this ability, cells derived from severalhuman tumors were incubated in the presence or absence of a glutathioneS-transferase-SDI-1 fusion protein. In the experiment, 5×10³ cells wereplated overnight at 37° C. (only for adherent cells) and then incubatedwith the SDI fusion protein. After 48 hours at 37° C., cells were pulsedwith thymidine for 24 hours and then harvested. The results of thisexperiment are shown in Table 8; the thymidine incorporation byuntreated cells was expressed as 100%. all determinations were made inquadruplicate.

TABLE 8 Antiproliferative Effects of SDI-1 Relative Cell Viability (% ofControl) Cell Line 50 μg/ml 30 μg/ml Myeloid Cells: Promyelocytic(HL-60) 1 ± 0 1 ± 0 Promonocytic (ML-1) 1 ± 0 1 ± 0 Myelogenous (KG-1) 1± 0 1 ± 0 Myelogenous (KG-1a) 1 ± 0 1 ± 0 Histiocytic Lymphoma (U-937) 1± 0 1 ± 0 Promonocytic (THP-1) 1 ± 0 1 ± 0 B Cell Lymphoma BurkittLymphoma (Daudi) 1 ± 0 3 ± 0 Burkitt Lymphoma (Raji) 1 ± 0 1 ± 0Epithelial Cells Breast (BT-20) 1 ± 0 1 ± 0 Breast (BT-20 TNF R) 1 ± 0 1± 0 Breast (SK-BR3) 1 ± 0 1 ± 0 Breast (MCF-7) 1 ± 0 1 ± 0 Breast (T-47D) 2 ± 0 2 ± 0 Lung adenocarcinoma (A-549) 25 ± 3  40 ± 1 Hepatocellular (Hep G2) 12 ± 2  21 ± 3  Glioblastoma Cells Glial (U-251)35 ± 2  66 ± 4  Normal Cells Human umbilical vein 1 ± 1 5 ± 1endothelial cells Human foreskin fibroblasts 1 ± 0 Not Done Murine TumorCells Fibroblasts (L-929) 4 ± 1 Not Done

EXAMPLE 14 Effect of SDI-1 cDNA on the Proliferation of Tumor Cells

The capacity of SDI-1 CDNA to repress the proliferation of tumor cellswas evaluated. SDI-1 cDNA was introduced into a number of tumor derivedand other cell lines by electroporation. One μg of the CMV-SDI-1 plasmidwas mixed with 1 μg of plasmid containing the CMV promoter and theβ-galactosidase gene. After electroporation, cells were plated and 24hours later assayed for the ability to incorporate tritiated thymidine.SDI-1 cDNA caused significant inhibition of DNA synthesis in a number oftumor derived cell lines including a melanoma, lung tumor and a braintumor. The SDI-1 cDNA also inhibited DNA synthesis in mouse 3T3 cellsand in normal bovine smooth muscle cells. Three tumor derived cell lines(one lung tumor cell line, and two kidney tumor cell lines) wereunresponsive to the SDI-1 cDNA.

EXAMPLE 15 Effect of SDI-1 Antisense cDNA on the Proliferation of NormalCells

The effect of SDI-1 on DNA synthesis was also evaluated using antisenseexpression vectors. SDI cDNA was provided to cells by introducingplasmid pCMVSDI684 which is a derivative of plasmid pCMVβ that lacks theβ-galactosidase gene, and which contains nucleotides 1-684 of the SDI-1cDNA sequence. Antisense vectors were constructed by cloning SDI-1antisense sequences into pMET, an inducible expression vector containingan altered human metallothionein promoter (CSIRO, BiomolecularEngineering, New South Wales, AU). The promoter contains a deletion inthe basal promoter and the addition of synthetic metal response elementsin triplicate. For construction of pMET, adenovirus sequences containingE1A gene 12S and 13S introns (i.e. nucleotides 917-1673) were firstcloned into the mammalian expression vector pRc/CMV (Invitrogen) at theNotI/ApaI sites of the multiple cloning region of this plasmid to createpRc/CMV-Ad. To ensure that no translation of E1A sequences occurred, andto create an SPEI cloning site in the vector, an SPEI linker containinga stop codon was inserted between the CMV promoter and the ElA splicesequence in-frame with the ElA sequence. The CMV promoter of plasmidRc/CMV-Ad was then replaced with the pM26 promoter to create pMET.

To accomplish this, the metallothionein promoter was excised from pM26by BgIII digestion and inserted into the BamHI site of pBluescript(Stratagene). An EcoRV and NotI fragment containing the promoter wasthen subcloned from pBluescript into a filled-in BgIII site and NotIsite of Rc/CMV-Ad. Antisense sequences from the SDI-1 cDNA were derivedfrom the full length cDNA cloned into the BamHI site of pBluescript. TheStuI site at nucleotide 127 was converted to a SpeI site by insertion ofan SpeI linker to make SDISPE127. An SpeI linker was also inserted atthe ApaI site at nucleotide 319 to create SDISPE319. These constructswere digested with SpeI and inserted in antisense orientation into theSpeI cloning site of pMET, thus creating the SDI antisense expressionvectors pMET-AS127 (having the first 127 nucleotides of the antisensestrand to SDI-1 cDNA, SEQ ID NO:1) and pMET-AS318 (having the first 319nucleotides of the antisense strand to SDI-1 cDNA, SEQ ID NO:1).

Cell lines expressing SDI-1 antisense were obtained by calcium phosphatetransfection. 3×10⁵ HCA2 cells (human foreskin cells) at populationdoubling 10 were transfected with 20 μg pMET, pMETAS127, or pMETAS318.Following two weeks of G418 selection, colonies were picked andexpanded. Inducibility and integrity of the inserted sequence wasdetermined using stable transformants by addition of 100 μM ZnCl₂ and 2μM CDCl₂ followed by RNA analysis of total cell RNA. The analysis was anRNase protection using antisense RNA probes, internally labeled with[³²P]-UTP. The probe pMET+SDI-1, containing SDI-1 nucleotides 444-686(of SEQ ID NO:1), was used to measure the expression of SDI-1 from boththe transfected genes and endogenous mRNA. After EcoNI digestion,transcription from the SP6 promoter in the pMET vector resulted in anantisense labeled probe that hybridized to both RNA from the expressionvector and to endogenous SDI-1 mRNA. This allowed a comparison ofrelative levels of expression from the introduced construct and SDI-1endogenous RNA. As a control, β-actin mRNA was measured in all assays.

In order to determine the extent of DNA synthesis, the cells weretrypsinized and 1×10⁴ cells were seeded per well in 24 well plates. Fourto six hours after plating, the cells were washed 3 times with phosphatebuffered saline and the medium was replaced with medium containing 0.5%fetal bovine serum. This serum deprivation induced the cells to becomequiescent. Six to ten days later, the medium was replaced and metal wasadded to induce the metallothionein promoter. The amount of metal addedwas optimized for each plasmid; optimized amounts were 70 μM ZnCl₂ and1.4 μM CdCl₂ for pMET1 and 50 μM ZnCl₂ and 1 μM CdCl₂ for AS1. Twentyhours later, induction was boosted by the addition of fresh medium.[³H]-thymidine (1.5 μCi/ml) was added to each culture 4 hours later.Twenty-four hours later, cells were fixed and analyzed byautoradiography. The results obtained in a typical experiment are shownin Table 9, and demonstrate the capacity of the antisense molecules ofthe present invention to induce the proliferation of quiescent cells.

TABLE 9 % of Cells Labeled Before Metal After Metal Cell Line InductionInduction No Integrant <5%  5% Vector Control <5%  5% AntisenseConstruct I  7% 49% Antisense Construct II <%5 57%

EXAMPLE 16 Abillity of SDI-1 Antisense Oligonucleodides to Repress theSDI-MEDIATED Ingibition of Proliferation

As discussed above, the expression of SDI-1 inhibits DNA synthesis. Insome instances, such as to immortalize human cells in culture, it isdesirable to repress such inhibition. The SDI-1 antisense molecules ofthe present invention are capable of mediating such repression.

In order to demonstrate the capacity of SDI-1 antisense oligonucleotidesto repress the DNA synthesis-inhibitory effects of SDI-1, cells wereprovided with an oligonucleotide having the sequence:

SEQ ID NO:3 AGCCGGTTCTGACATGGCG

This oligonucleotide is antisense to SEQ ID NO:1 at nucleotides 75-93.

Cells were cultured in medium containing 10% fetal bovine serum (FBS)for approximately one week, at which time the medium was replaced withmedium containing 0.5% FBS. The cells were divided into control cellsand experimental cells. On Day 1, experimental cells were provided witheither 1, 2 or 5 μM of the above-described oligonucleotide; Controlcells received no oligonucleotide. On Day 3, and again on Day 5, allcells received fresh medium containing 0.5% FBS; experimental cellsreceived additional oligonucleotides (1, 2 or 5 μM). On Day 6, tritiatedthymidine was added to the culture media and the cells were harvested onday 7. Experimental cells (that had received the antisenseoligonucleotide) exhibited an increase in the amount of tritiatedthymidine incorporated into their DNA, relative to the control cells.

The results indicated that oligonucleotides that contain regionscomplementary to SDI-1 nucleotides 75-93 can act as antisense repressorsof SDI-1 function.

EXAMPLE 17 Effect of DNA Damage and Growth Arrest on the Expression ofSDI-1

The above-described experiments demonstrated that p53 induces SDI-1expression, and that the production of SDI-1 mRNA was induced incontact-inhibited and serum-deprived cells, as well as in senescenthuman cells. To determine whether the induction of SDI-1 was a generalcharacteristic of growth arrest states, the effect of DNA damage onSDI-1 mRNA levels was evaluated.

Cells are believed to undergo growth arrest in response to DNA damagingagents before entering the S or M phases of the cell cycle. The growtharrest permits the cell to repair any genetic lesions caused by the DNAdamaging agents. A failure to repair such damage creates a mutation, andmay have severe consequences ranging from cell death to neoplasia. Thus,the capacity of a cell to undergo growth arrest in response to DNAdamage has great importance both in the etiology of cancer and in theresponse of cancer cells to chemotherapy.

To evaluate the effect of DNA damaging agents on SDI mRNA production,normal human neonatal foreskin fibroblast cells (strain HCA2); animmortalized cell line (TE85, obtainable from the American Type CultureCollection, Rockville, Md., US), and the above-described MDAH 041 celllines were employed. All cells were cultured in Eagle's minimalessential medium with Earl's salts plus 10% fetal bovine serum (GibcoBRL, Gaithersburg, Md., US) in 5% CO₂ at 37° C., or in Hanks salts plus10% fetal bovine serum at 37° C. HCA2 cells achieve approximately 80population doublings before becoming senescent. The cells were used atpopulation doubling 27 or less.

Cells were plated at 1×10⁴ cells/cm² on glass coverslips and grown 24-48hours to 50-75% confluency and were then treated with one of several DNAdamaging agents: γ-rays (4 Gy); bleomycin (75 μg/ml; 4 hours), etoposide(400 μM in 4% DMSO; 8 hours), hydrogen peroxide (400 μM; 1 hour), UVlight (30 J/m²), methyl methane sulfonate (“MMS”) (100 μg/ml; 4 hours),mitomycin C (“MMC”) (5 μg/ml; 30 hours) and CdCl₂ (250 μM; 1 hour). ForUV irradiation, cells were cultured in 150 cm² tissue culture dishes.Immediately prior to treatment the medium was removed and the dishes,without lids, were placed in a Stratagene (La Jolla, Calif., US)StratalinkerUV cross-linking device and irradiated with a dose of 30J/m². Fresh serum-supplemented medium was then added and the cells wereincubated at 37° C., 5% CO₂. γ-irradiation was performed on cells incomplete cell culture medium in 25 cm² tissue culture flasks, using afixed ¹³⁷Cs source. The dose rate was 4.21 Gy/min. Exposure was to 4 Gy.In addition, the effects of heat shock (42° C.; 4 hours), hydroxyurea (2mM; 24 hours) and prostaglandin A₂ (10 μg/ml in 0.1% ethanol; 24 hr)were evaluated.

Tritiated thymidine was added 16 hours following each such treatment andthe cells were incubated for 8 hours, fixed and processed forautoradiography as described above. RNA was harvested 4 or 24 hours posttreatment using RNAzol B (Cinna/Biotecx, Houston, Tex.) according to themanufacturer's protocol. 10-20 μg of RNA was separated on a 1.2% agarosegel with 20% formaldehyde, transferred to a Gene screen plus (EN,Boston, Mass.) membrane, probed, and quantitated. Fold induction wasdetermined by comparing treated samples with untreated controls.Controls were mock treated cells put through the same washing, mediachange, transportation, and inadvertent temperature fluctuation as thosereceiving treatment. Controls for agents dissolved in ethanol ordimethyl sulfoxide were exposed to the same concentration of the solventalone. SDI-1 RNA values were normalized to GAPDH levels. In some casesnormalization to actin RNA was also done and revealed no substantialdifferences in fold induction. The results of this experiment are shownin Table 10.

TABLE 10 Induction of SDI-1 mRNA and Growth Arrest by Various TreatmentsMechanism SDI-1 % Growth of Action Agent Dose Level* ArrestDouble-Stranded γ-ray 4 Gy 2.0  70% Breaks Double-stranded bleomycin 75μg/ml 11.8 100% Breaks 4 hr Protein Associated etoposide 400 μM 8.0 100%Double-Stranded 8 hr Breaks Free Radicals H₂O₂ 400 μM 6.3  95% 1 hrBulky Adducts UV light 30 J/m² 15.2  60% (some free radicals) AlkylationMMS 100 μg 3.27  53% 4 hr Cross-Linking Mitomycin C 5 μg/ml 2.8 100% 30hr Mutagenic Metal CdCl₂ 250 μM 1.3  0% 1 hr General Stress Heat Shock42° C. 2.3 Not Done 4 hr Inhibitor of dNTP hydroxyurea 2 mM 7.9 100%Synthesis 24 hr Paracrine Hormone prostaglandin A₂ 10 μg/ml 6.1 100% 24hr *Fold induction of SDI-1 at 24 hour level

Preliminary studies (EI-Deiry, W. S. et al., Cell 75:817-825 (1993))suggested that the production of WAF1 might be induced by UV light. Theabove experiments demonstrate that the production of SDI-1 mRNA isinduced by DNA damage and other growth arrest treatments in both normalcells and cells that lack wild type p53. The results of theabove-described experiment demonstrate the role of SDI-1 in the repairof DNA damage or mutations that could lead to neoplasia in mammaliancells.

Every agent studied, with one exception, was found to cause an increasein SDI-1 mRNA in these cells (Table 10). Cadmium chloride, at theconcentration used, failed to increase SDI-1 message level. However,tritiated thymidine incorporation analysis revealed that the cadmiumchloride treatment did not induce growth arrest (no inhibition of DNAsynthesis) (Table 10). In contrast, growth arrest, as measured bytritiated thymidine incorporation, was essentially complete in responseto the other agents tested (Table 10). However, in the case of UVirradiation and MMS treatment, an inhibition of only 50-60% wasobserved. It is possible that growth arrest, in response to theseagents, had not reached its maximum at the time DNA synthesis wasmeasured or conversely that cell proliferation had already resumed. Thelack of induction by cadmium chloride indicates that SDI-1 message wasincreased by DNA damage only when the damage caused growth arrest. Thisis supported by the observation that hydrogen peroxide was unable tofurther elevate SDI-1 mRNA levels in cells that were already growtharrested by contact-inhibition.

The kinetics of SDI-1 RNA induction by γ-irradiation and hydrogenperoxide treatment was found to differ (FIG. 6), providing furtherevidence for multiple mechanisms involved in regulating SDI-1 geneexpression. Induction of SDI-1 message by γ-irradiation follows a timecourse for this gene by p53. It has been demonstrated in mouse prostatecells that p53 activity increases rapidly (within 30 minutes) followingexposure to ionizing radiation and then declines to baseline levelwithin 3 hours (Lu, X. et al., Cell 75:765-778 (1993)). The induction ofSDI-1 mRNA observed in normal human cells is similar to this patternthough lagging in time slightly as would be expected. The majordifference is that SDI-1 message declined more slowly (overapproximately 30 hours) than p53 activity, thus explaining theobservation of Lu, X. et al. (Cell 75:765-778 (1993)). The induction ofSDI-1 mRNA observed in normal human cells is similar to this patternthough lagging in time slightly. The major difference is that SDI-1message declined more slowly (over approximately 30 hours) than p53activity. This explains the observation of Lu, X. et al. (Cell75:765-778 (1993)), that ionizing radiation-induced arrest lasts for20-30 hours, long after p53 activity had declined.

Induction of SDI-1 message by hydrogen peroxide (FIG. 6) exhibited avery different pattern. It has been proposed that exposure to hydrogenperoxide results in a rapid increase in p53 activity (Tishler, R. B. etal., Canc. Res. 53:2212-2216 (1993)), which returns to baseline by 4hours post-treatment. SDI-1 mRNA level, in contrast, did not risesignificantly until 9 hours after treatment and continued to increasefor at least 48 hours (FIG. 6). The slow response of SDI-1 RNA level tohydrogen peroxide treatment indicates that while an elevated level ofSDI-1 mRNA appears to be universally associated with growth arrest, itmay not always be the initial cause of the arrest. A similar pattern ofSDI-1 message elevation was observed by serum-deprivation andcontact-inhibition of normal human cells and resembles what is observedin cases of mRNA stabilization. A slow increase of mRNA in response toserum-deprivation and contact-inhibition has also been noted for theGADD genes (Fornace, A. J. et al.,Molec. Cell. Biol. 9:4196-4203(1989)). In all these cases, SDI-1 may act to maintain growth arrestafter it has been initiated by another pathway.

EXAMPLE 18 Role of P53 in the SDI-1 Response to DNA Damage and GrowthArrest

As indicated, agents that cause growth arrest but do not damage DNA(heat shock, hydroxyurea, and prostaglandin A₂) were also examined(Table 10) and found to cause an increase in SDI-1 message levels. Heatshock and hydroxyurea treatments increased SDI-1 message (Table 10)though both have been shown not to increase p53 activity (Lu, X. et al.,Cell 75:765-778 (1993); Zhan, Q. et al., Molec. Cell. Biol. 13:4242-4250(1993)), indicating these agents may operate through a p53-independentmechanism.

In order to further investigate this possibility, the p53 status ofvarious immortal cell lines was determined through immunoprecipitationstudies and compared to the steady state level of SDI-1 mRNA in the samecell lines. For this purpose, cell lines were grown to approximately 70%confluence in 75 cm² flasks prior to being cultured for 1 hour inmethionine-free modified Dulbecco's Eagle medium, and then labeled inmethionine-free medium containing 10% dialyzed fetal bovine serum and100 μCi/ml L-[³⁵S] methionine (TRAN35SLABEL; ICN). One flask was usedper immunoprecipitation. After a 3 hour incubation in this labelingmedium, the cells were placed on ice, washed with ice-cold phosphatebuffered saline (PBS) and harvested in lysis buffer (20 mM Tris, pH 7.4,150 mM NaCL, 0.5% NP-40, 1 mM EDTA, 50 μg/ml leupeptin, 30 μg/mlaprotinin). Lysates were scraped into microfuge tubes and sheared 3times by passage through a 25 gauge needle. Phenylmethylsulfonylfluoride was added to a final concentration of 1 mM and lysatescentrifuged at 14,000 r.p.m. in an Eppendorf microfuge at 4° C. for 45min. Supernatant volumes were adjusted to normalize for cell number,then precleared for 30 minutes at 4° C. for 45 min. Supernatant volumeswere adjusted to normalize for cell number, then precleared for 30minutes at 4° C. on a rotating wheel with approximately 40 μl packedvolume of protein A-agarose (Pharmacia) that had been sequentiallycoated with rabbit anti-mouse IgG (ICN) and a mixture of two mouse IgG2amyeloma proteins (ICN). Lysates were then microfuged and each resultingsupernatant was incubated with 50 μl packed volume of protein GPLUS/Protein A-agarose (Oncogene Science) coated with 4 μg of anappropriate p53-specific antibody or a nonspecific antibody of the sameclass and subclass. Specific antibodies were PAb421, which binds bothwild-type and mutant forms of P53, PAb 240 which recognizes a subset ofmutant forms, and PAb1620, which recognizes wild type p53 specifically.All p53 antibodies were obtained from Oncogene Science. Non-specificantibodies used were either a non-specific IgG₁ antibody to E. colianthranilate syntheses protein or a mixture of two IgG_(2a) myelomaproteins. Incubations were carried out for 2 hours at 4° C. on arotating wheel. Samples were microfuged and pellets washed 4 times withice-cold wash buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 0.5% NP-40, and0.1% SDS) then 2 times with PBS. Samples were resuspended in 2× loadingbuffer (4% SDS-polyacrylamide gel, pH 8.8. The gel was fixed, treatedwith Amplify (Amersham), dried and exposed to Kodak X-AR film for 20-28hours at 80° C.

If p53 had been the only regulator of SDI-1 mRNA transcription, theabsence of wild type p53 would have coincided with a low level of SDI-1expression, whereas, the presence of functional p53 should havecorresponded to a higher level of SDI-1 expression.

Evidence for a p53-independent mechanism of regulation of SDI-1expression was obtained by exposing TE85 and MDAH 041 cells, which lackfunctional p53, to many of the agents described in Table 10 anddetermining changes in SDI-1 message levels. TE85 is an osteosarcomaderived line, which has been found, by imunoprecipitation, to expressonly mutant p53. All treatments examined except γ-irradiation, whichrequires wt p53 for G₁ arrest (Kuerbitz, S. J. et al., Proc. Natl. Acad.Sci. USA 89:7491-7495 (1989)), were able to increase SDI-1 message inthe cell line TE85 (Table 11). The p53-independent upregulation of SDI-1RNA level was confirmed in MDAH041 cells following hydrogen peroxide andhydroxyurea treatment (Table 11). The pattern of SDI-1 mRNA accumulationis therefore similar to that of GADD45. This gene also requiresfunctional p53 for induction by γ-irradiation but not by most otheragents (Zhan, Q. et al., Molec. Cell. Biol. 13:4242-4250 (1993)). GADD45is a member of a family of genes that were identified based upon theirinduction by both growth arrest and DNA damage (Fornace, A. J. et al.,Molec. Cell. Biol. 9:4196-4203 (1989)). These findings indicate that P53is not the only regulator of SDI-1, and that additional regulators ofSDI-1 exist. The above methods can be used to identifiy such additionalregulators.

TABLE 11 Agent TE85 MDAH 041 Control 1 1 γ-rays 1.3 1.1 etoposide 2.6H₂O₂ 4.0 3.3 UV light 3.0 MMS 4.4 MMC 2.5 Hydroxyurea 7.5 2.5

In summary, the above results demonstrate that: 1) a wide variety ofagents that induce growth arrest, including those that damage DNA,elevate SDI-1 message level; 2) some agents can accomplish this in theabsence of functional P53 and 3) this elevation follows two distincttemporal patterns. In one, a rise in p53 activity results in a parallelincrease in SDI-1 mRNA. This occurs early enough to be causative ofarrest. In the second, an inhibitor of growth causes arrest by amechanism that does not depend entirely upon p53. This arrest isassociated with a gradual accumulation of SDI-1 mRNA, perhaps due to anincrease in message stability.

EXAMPLE 19 Production of Monoclonal Antibodies to SDI-1 Fusion Proteins

Anti-SDI-1 monoclonal antibodies were isolated as described above usingthe GST-SDI-1 fusion as an immunogen and using the [His]₆ fusion havingthe leader sequence of SEQ ID NO:4 in a screen for suitable hybridomas.All of the antibodies could immunoprecipitate both cellular andrecombinant SDI-1 protein in addition to the GST-SDI-1 fusion protein.

The monoclonal antibodies were used to in an imunohistochemicalevaluation of human tissue. Antibodies (10 μg/ml) from four hybridomalines (18A10; C6B6; 8A8; 2G12), and from a mouse IgG_(2b) control, wereincubated with neat supernatant overnight at 4° C. Antibody was detectedusing the Vector Standard ABC kit with DAB as chromogen. Tissue fromhuman tonsil (containing both a lymphoid and an epithelial cellcomponent), adult human skin (containing both connective tissue andepithelial cell components) or adult human colon (containing connectivetissue and glandular epithelial cell components) was evaluated. Stainingwas evaluated on a qualitative +/− scale. The control antibody failed toelicit staining on any tissue.

Monoclonal Antibody 18A10

Tonsil all epithelial cell layers, all lymphocytes and all stromalcomponents did not stain.

Skin all upper layers and possibly the basal cell layer showed a traceto 1+ diffuse cytoplasmic staining. The connective tissue components didnot stain.

Colon Epithelial cells did not stain. The mucous within the glandsexhibited a trace to 1+ staining. The connective tissue components didnot stain.

Monoclonal Antibody C6B6

Tonsil all epithelial cell layers, all lymphocytes and all stromalcomponents did not stain.

Skin basal cell epithelial cells had a +/− to trace+ diffuse cytoplasmicstaining. The connective tissue components did not stain.

Colon Individual cells among the epithelial cells showed a 3+nuclear/cytoplasmic staining. The mucous within the glands did notstain. The connective tissue components did not stain.

Monoclonal Antibody 8A8

Tonsil all upper layer epithelial cell layers and possibly basal cellsshowed a +/− to trace+ diffuse cytoplasmic staining. Lymphocytes and allstromal components did not show immuno-reactivity.

Skin all upper epithelial cells showed a +/− to 1+ diffuse cytoplasmicstaining. The connective tissue components did not stain.

Colon Areas of epithelial cells showed a 3+ nuclear/cytoplasmicstaining. The mucous exhibited a +/− staining. The connective tissuecomponents did not stain.

Monoclonal Antibody 2G12

Tonsil the basal cell epithelial layer showed a trace to 1+ staining.Some lymphocytes exhibited a trace membrane staining. The stromalcomponents did not stain.

Skin the basal cell layer showed a trace to 1+ diffuse cytoplasmicstaining. The upper cell layers did not appear to stain.

Colon Significant brown precipitated material was evident, suggestingthat some ductal epithelail cells were exhibiting a strong nuclearstain. The mucous within the glands did not stain. The connective tissuecomponents did not stain.

The experiment indicated that the monoclonal antibodies coulddistinguish between tissue types that expressed SDI-1 and those that didnot express SDI-1. Moreover, it was possible to discern a differentialpattern of staining in different cell types. The immunohistochemicalevaluation could be performed on biopsied tissue. The detection ofnon-staining foci of cells within the tumor mass would be indicative ofcells that no longer expressed SDI-1, and which therefore would warrantmore aggressive antineoplastic and antimetastatic therapy.

EXAMPLE 20 Delivery of SDI-1 to Target Cells

The ability to deliver a pharmacological agent to a cell constitutes ageneral problem in defining therapeutic protocols. In order to evaluatethe role of the GST moiety in facilitating the cellular uptake of SDI-1,the drug-delivery capacity of two GST-SDI-1 constructs was evaluated.

Construction of the First GST-SDI-1 Fusion

The first of these fusions was the especially preferred Schistosomajaponicum GST-SDI-1 fusion (discussed above) that had been initiallyrecognized as being capable of entering target cells. Remarkably,however, slight modifications in the structure of this fusion were foundto substantially destroy its capacity to be taken up by target cells.Thus, the originally described Schistosoma japonicum GST-SDI-1 fusionhad inherent and distinctive characteristics that were not previouslyfully recognized.

The especially preferred Schistosoma japonicum GST-SDI-1 fusion wasformed by cleaving plasmid pcDSRαΔ-SDI-1 with Acyl and EcoRI, therebyliberating an SDI-1-encoding fragment. Acyl cleaves the plasmid 4nucleotides preceding the SDI-1 initiation codon; EcoRI cleaves themolecule at approximately position 1010 of SEQ ID NO:1. The fragment wasthen cloned between the Smal and EcoRI sites of plasmid pBS. Thisconstruct was then cleaved with BamHI and EcoRI to release anSDI-1-encoding fragment that contained the nucleotides: GGATCCCCCCGCC(SEQ ID NO:7) immediately preceding the SDI-1 ATG initiation codon. Thedistal terminus of the molecule was at approximately position 1010 ofSEQ ID NO:1 .

Plasmid pGEX2T, which contains the Schistosoma japonicum GST-encodingsequence was cleaved with BamHI and EcoRI. The BamHI enzyme cleaves theSchistosoma japonicum GST-encoding sequences at a site located atnucleotides 662-667 of the molecule (approximately at codon 226 of themolecule); the EcoRI enzyme cleaves the plasmid outside of theGST-encoding sequences. The dual enzymatic cleavage prevents religationof the plasmid, and ensures that the SDI-1 sequences will be introducedin the proper orientation.

The BamHI-EcoRI treated pGEX2T DNA was incubated with theabove-described SDI-1-encoding fragment that contained, at one end thenucleotides: GGATCCCCCCGCC (SEQ ID NO:7) immediately preceding the SDI-1ATG initiation codon, and at the other end an EcoRI cleavage site.Ligation of the two molecules produced a plasmid in which theSDI-1-encoding sequences were fused one nucleotide out of frame from theGST-encoding sequences. In order to restore reading frame, the plasmidwas cleaved with BamHI, the staggered ends were filled in using KlenowDNA polymerase, and then ligated to a 10 base pair Xhol linker (Promega)having the sequence: CCCTCGAGGG (SEQ ID NO:8). Thus, the final constructcontained sequences encoding amino acids 1-226 of GST fused to a 9residue fragment that encoded Pro-Arg-Gly (residues 1-3 of SEQ ID NO:9)fused to a BamHI-filled linker (SEQ ID NO:7) that encodedAsp-Pro-Pro-Ala (residues 4-7 of SEQ ID NO:9) fused to the ATGinitiation codon of SDI-1 and the remainder of the SDI-1-encodingsequence. The nucleotide sequence of the complete gene fusion isprovided in SEQ ID NO:10; the amino acid sequence encoded by this genefusion is provided in SEQ ID NO:11. An E.coli strain transformed withplasmid pAG20, containing the above-described first GST-SDI-1 genefusion was deposited with the American Type Culture Collection(Rockville, Md., U.S.) under the terms of the Budapest Treaty governingmicrobial deposits. The deposit was made on Apr. 5, 1994, and was giventhe Deposit Accession Number ATCC 69597.

Construction of the Second GST-SDI-1 Fusion

The second SDI fusion was obtained by amplifing the SDI-1 coding regionof plasmid pcDSRαΔ-SDI-1 using the polymerase chain reaction, andprimers having the sequence:

Primer 12614 (SEQ ID NO:12) GGAGGATCCATGTCAGAACCGGCT

Primer 12615 (SEQ ID NO:13) GCAGAATTCCTGTGGGCGGATTAG

These primers amplify a polynucleotide containing residues 77-587 of SEQID NO:1. Primer 12614 has a BamHI site immediately upstream of the ATGtranslation initiation site. Primer 12615 includes 14 nucleotidesdownstream of the translational stop site, and includes a partial EcoRIsite.

The amplified product was cleaved with BamHI and EcoRI and cloned intothe above-described BamHI/EcoRI treated pGEX2T plasmid. The resultingplasmid contained an in-frame fusion linking codon 226 of the GSTsequence to codon 1 of SDI-1.

The two protein fusions thus differ only in that the first fusioncontains a “hinge region” consisting of the amino acids:

SEQ ID NO:9 Pro-Arg-Gly-Asp-Pro-Pro-Ala

whereas the second region does not contain any hinge region.

When the first and second GST-SDI-1 fusions were provided to youngcells, the first fusion protein was found to be detectable within thecells whereas the second fusion protein was not detected within thecells. These observations indicated that whereas the first GST-SDI-1fusion had the capacity to bind to cells and to be incorporated intorecipient cells, the second GST-SDI-1 fusion lacked this capability.Consistent with these observations, of young cells that had been exposedto either the first protein fusion or the second protein fusion, onlycells exposed to the first protein fusion exhibited quiescence.

EXAMPLE 21 Deletion Analysis of SDI-1 Protein

In order to investigate the functional domains of the SDI-1 protein,several genetic constructs were prepared in which the CMV promoter wasused to drive transcription of fragments of the SDI-1 cDNA sequence.

Specifically, SDI-1 polynucleotides were constructed that lacked codonsfor amino acids: 24-29 (designated as: SDI-1_(1-23;25-164)); 30-35(designated as: SDI-1_(1-29;36-164)); 42-47 (designated as:SDI-1_(1-41;48-164)); 53-58 (designated as: SDI-1_(1-52;59-164)); 66-71(designated as: SDI-1_(1-65;72-164)); 72-164 (designated as: SDI-1₁₋₇₁).In addition, an SDI-1 polynucleotide was constructed that had beentreated to remove a Stu-Tth fragment that included codons 16-52(designated as: SDI-1_(Stu-Tth)). These constructs, and an intactSDI-1-encoding construct (designated as: SDI-1₁₋₁₆₄) and a CMV controlvector were co-transfected into MDAH 041 cells along with a vector thatexpressed β-galactosidase driven by the CMV promoter. The percent ofgrowth inhibition was determined relative to cells that had beenco-transfected with the CMV-β-gal and CMV vector. Experiments were donein triplicate. The percent inhibition obtained for each SDI-1polynucleotide is shown in Table 12.

TABLE 12 Amino Percent Inhibition Acids Expt. Expt. Expt. ConstructDeleted 1 2 3 Avg. CMV-vector none 98 100 98 99 SDI-1_(1-23;25-164)24-29 93 93 92 93 SDI-1_(1-29;36-164) 30-35 93 91 83 89SDI-1_(1-41;48-164) 42-47 66 76 75 72 SDI-1_(1-52;59-164) 53-58 40 46 5748 SDI-1_(1-65;72-164) 66-71 88 81 88 86 SDI-1_(Stu-Tth) Stu-Tth 11 3613 20 SDI-1₁₋₁₆₄  72-164 90 98 98 95

The results indicate that deletion of amino acids 42-47 caused adecrease in the efficiency and extent of inhibition. Deletion of aminoacids 53-58 showed a major decrease in efficiency and extent ofinhibition. Deletion of the carboxy terminal portion of the SDI-1molecule (amino acids 72-164) did not significantly affect the extent ofinhibition. Thus, active domains of SDI-1 are present within a peptidefragment containing amino acids 1-71, and amino acids 42-47 and 53-58contain active SDI-1 domains. In sum, active domains of SDI-1 arecontained between amino acids 42-58 of the SDI-1 protein.

EXAMPLE 22 The Identification of an Immunoreactive 23 KD Protein

As expected, all of the above-described monoclonal and polyclonalantibodies were found to be capable of immunoprecipitating a 21 kDprotein (“p21”) corresponding to SDI-1. Analysis of the proteinimmunoprecipitated by these antibodies revealed that, unexpectedly, theantibodies also precipitated a 23 kD protein that was present incellular extracts. The fact that all of the anti-SDI-1 antibodies testedprecipitated this 23 kD protein (“p23”) indicates that SDI-1 and the 23kD protein are structurally related and share multiple epitopes. Thefact that the 21 kD SDI-1 protein expressed from the above-describedcDNA vectors is biologically active, indicates that the 23 kD proteinexhibits the characteristics of an inactive, phosphorylated precursor ofthe active 21 kD SDI-1 protein.

In possessing such a precursor, SDI-1 resembles the Rb protein which hasbeen found to exhibit multiple phosphorylation states that havemolecular weights of 110-114 kD; the dephosphorylated form has amolecular weight of 110 kD (Lee, W.-H. et al., In: Tumor SuppressorGenes, Klein, G. (ed.), Marcel Dekker, Inc., New York, pp. 169-200(1990)).

The identification of an inactivated, phosphorylated form of SDI-1 wouldindicate that the biological activity of SDI-1 is both positivelyregulated by p53 induction of transcription and negatively regulated bya cellular kinase. The identification of such kinase(s) can be readilydetermined by screening a cDNA library for members that upon expressionproduce proteins that can increase the phosphorylation ofdephosphorylated p21 SDI-1. Such conversion can be analyzed byconducting western blots of p21-p23 immunoprecipitated protein. Sincesuch enzymes inactivate p21 SDI-1, they confer a proliferative capacityto the cell, and can be identified as molecules that overcomeSDI-1induced senescence. Such molecules can be employed in the samemanner as SDI-1 antisense nucleic acids. Similarly, enzymes thatdephosphorylate the p23 protein can be identified by screening forpolynucleotides that, upon expression, form a protein that can convertthe p23 form into the p21 form. Western blot methods can be used toidentify such molecules. Since such enzymes convert the p23 moleculeinto the active p21 form, such enzymes confer a quiescent oranti-proliferative capacity to the cells. Such molecules can be employedin the same manner as SDI-1 or SDI-1 encoding nucleic acids.

EXAMPLE 23 Characterization of SDI-1 Fragmants

As discussed above, the control of cell proliferation in eukaryotes fromyeast to humans involves the regulated synthesis, activation, anddegradation of a family of cyclins that act as the regulatory subunitsof protein kinases termed cyclin-dependent inases (Cdks). The cdks arenecessary for the start of S phase and mitosis. The mechanism ofinhibition of DNA synthesis by the protein SDI-1 involves inhibition ofa series of Cdk kinase activities (e.g., cdk1-cdk6)). Indeed, in vivoSDI-1 protein has been found to associate with several of thesecdk-cyclin complex by immunoprecipitation with antibodies against thevarious cyclin antibody (see, Xiong, H. et al., Cell 71:505-514 (1992)).These complexes are disrupted upon cellular transformation with some DNAtumor viruses (PCT Patent Application WO94/09135; Waga, S. et al.,Nature 369:574-578 (1994)), confirming the above-stated mechanism bywhich oncogenic proteins alter the cell cycle of transformed cells. Inaddition, a recent report has confirmed the above-described relationshipbetween SDI-1 and p53, and has shown that wild type p53 directlytransactivates SDI-1 (Harper, J. W. et al., Cell 75:805-816 (1993);El-Deiry, W. S. et al., Cell 75:817-825 (1993); Xiong, Y. et al., Nature366:701-704 (1993); Dulic, V. et al., Cell 76:1013-1023 (1994)), thusconfirming that it is the downstream effector of p53. These resultsconfirm that SDI-1 is not only a negative regulator during normal cellproliferation but also a tumor suppresser factor during carcinogenesis.

In order to determine the region(s) of the SDI1 molecule that wasrequired to inhibit DNA synthesis, a series of plasmids was constructedin which various SDI-1 cDNAs, expressed from the CMV promoter, wereprogressively truncated from the 3′ end of the coding region. Thecapacity of the deletion mutants to inhibit kinase activity and/or DNAsynthesis was evaluated using a transient expression assay and bidingassay with cdk2.

The plasmids were constructed by digesting the plasmid pCMVβ with Notlto remove the E. coli β-galactosidase gene. This site was blunted withKlenow and an Spel linker was inserted in order to create pCMVSpel. Thefull-length SDI-1 cDNA was digested with BamHI and DraI and cloned intoa pBluescript vector. An Spel linker was litigated into the Hinc II siteof this vector to create Spel ends for nucleotides I-686 of the SDIIcDNA [plasmid pBSsdil(1-686)Spel]. The Spel-bounded fragment of SDI-1from this vector was then ligated into the pCMVSpel vector to createpCMVsdil(1-686), containing the full 164 amino acid coding region (SEQID NO:2) for the wild-type SDI-1 cDNA sequence. A series of mutantsencoding carboxy-terminal truncated versions of SDI-1 were generatedusing restriction enzymes that have unique sites within the SDI-1 codingsequence.

HCA2, the above-described normal human diploid fibroblasts derived fromneonatal foreskin were employed in the mutational analysis. These cellsachieve 80 population doublings (PD) before entering senescence and wereused at PD of 20-30 in all experiments. Each construct was tested forthe ability to inhibit the initiation of DNA synthesis when transfectedinto HCA2. Following transfection, the deletion constructs were testedfor protein production by immunofluorescence using an anti-HA monoclonalantibody (12CA5, obtained from BabCO).

Transfection of the full length SDI1 coding region resulted in a 90%decrease in the percentage of cells incorporating tritiated thymidine. Atruncated SDI-1 encoding only the amino terminal 71 amino acids was aseffective as the full length 164 amino acid protein in blocking entryinto S phase whereas, a more severely truncated form encoding only thefirst 52 amino acids was completely inactive (Table 13). This indicatedthat a critical region for the DNA synthesis inhibitory activity waslocated between amino acids 52 and 71. Thus, peptide fragments of SDI-1having amino acid residues 52-71 comprise mimetics of SDI-1 protein.Similarly, molecules that mimic a reactive side group of SDI-1₅₂₋₇₁comprise mimetics of SDI-1. The role of the amino terminal portion ofthe protein in inhibiting entry into S phase was further clarified whena construct with a deletion in amino acids 16-52 (plasmid “1-164(Δ16-52)”) was also found to have lost all DNA synthesis inhibitoryactivity.

TABLE 13 Amino Acid Residues Present % Inhibition ot DNA Synthesis InSDI-1 protein fragment Relative to Control 1-164 88% 1-123 90% 1-82  92%1-71  85% 1-52   0% 1-16   0% 1-164 (Δ16-52)  0%

To more finely map the DNA synthesis inhibitory region of the molecule,small internal deletions (in which only 4 to 6 amino acids were removedand 3 amino acids (Pro-Arg-Gly) were substituted) were introduced intothe amino terminal portion of the molecule that the large deletionconstructs had indicated was necessary for the inhibitory activity.Deletions of six amino acids were constructed in the SDI-1 cDNA at aminoacids 24-29, 30-35, 42-47, 53-58 and 66-71. These constructs were fusedin frame with DNA encoding a portion of the hemagluttinin (HA) moleculeto provide an in-frame hemagluttinin (HA) tag at the C-terminus of thefull length SDI-1 protein. The HA tag provided a unique antigen forimmunostaining, and permitted a determination of whether the proteinproducts were expressed and where they were localized within the cell.To ensure that the HA tag did not interfere with the DNA synthesisinhibitory activity of SDI-1, the cDNAs encoding either the full length(164 amino acid) or the truncated (amino acids 1-71) species were alsofused to the HA sequence at the carboxy terminus. The HA tag sequencewas introduced by the polymerase chain reaction (PCR) using the primers:

SEQ ID NO:14 TCTAGGCCTGTACGGAAGTG (splice site in pCMV vector) SEQ IDNO:15 TAGGAATTCACTAGTCTAAGCGTAATCTGG AACATCGTATGGGTAGGGCTTCCTCTTGGA

The inhibitory activity of these cDNAs was the same as that ofconstructs without the HA tag. Another control construct was deleted inamino acids 16-52 and had no inhibitory activity whether it was taggedor not. Interestingly, and in further support of the importance of theintegrity of the amino terminal region of the molecule in growthinhibition, cDNAs tagged with HA at the amino terminus had no growthinhibitory activity.

Deletions were then introduced into the protein-coding region of theSDI-1 cDNA in plasmid pCMVsdil(I-686)HA by PCR. The tag allowed one toascertain that the wild-type and mutated proteins were expressed incells transfected with the various constructs and also to determine thelocalization of the mutant protein.

The following primer sets were used to introduce the indicated deletions(Table 14):

TABLE 14 Amino Acids Seq De- Id leted No Nucleotide Sequence 24-29 16TTCGGCCCTCGAGGCCTGAGCCGCGACTGT 17 GCTCAGGCCTCGAGGGCCGAAGAGGCGGCG 30-3518 TTAGCGCGCCTCGAGGCTGCTCGCTGTCCAC 19 CGAGCAGCCTCGAGGCGCGCTAATGGCGGGC42-47 20 GGCTGCCCTCGAGGCCGATGGAACTTCGAC 21CCATCGGCCTCGAGGGCAGCCCGCCATTAG 49-53 22CGTGAGCGACCCCGGGGCGTCACCGAGACACCACTG 23CTCGGTGACGCCCCGGGGTCGCTCACGGGCCTCCTG 53-58 24TTCGACCCTCGAGGCCTGGAGGGTGACTTC 25 CTCCAGGCCTCGAGGGTCGAAGTTCCATCG 58-6126 ACCGAGACATCCCGGGCCGACTTCGCCTGGGAGCGT 27GGCGAAGTCGGCCCGGGATGTCTCGGTGACAAAGTC 61-66 28CCACTGGAGCCCCGGGGCCGTGTGCGGGGCCTTGGC 29CCGCACACGGCCCCGGGGCTCCAGTGGTGTCTCGGT 66-71 30GCCTGGCCTCGAGGCGGCCTGCCCAAGCTC 31 CAGGCCGCCTCGAGGCCAGGCGAAGTCACC 72-7732 CGGGGCCTTCCCCGGGGCCTTCCCACGGGGCCCCGGCGAGG 33CGTGGGAAGGCCCCGGGGAAGGCCCCGCACACGCTCCCAG

Each primer was used with a primer hybridizing within the vector toamplify a portion of the SDI-1 plasmid, the amplified materials from tworeactions were pooled and permitted to anneal. Single-stranded regionswere filled in with polymerase, and the fragments were then ligatedtogether to produce the desired convalently closed circular vectors. Theuse of SEQ ID NO: 26-27replaced SDI-1 amino acids 58-61 with thetripeptide “Ser-Arg-Ala.” The truncated constructs thus encoded theamino terminal fend of the protein up to amino acid 123, 82, 71, 52 and16, respectively. All constructs were sequenced to verify that thedesired deletions had been made, that the integrity of the HA tag wasintact and to confirm that no additional mutations had been introducedinto the constructs.

HCA2 and MDAH 041 cells were co-transfected with pCMVβ-gal and plasmidscarrying the above-described SDI-1 mutated DNA using calcium phosphateprecipitation. The MDAH 041 cell line was derived from a Li-Fraumenisyndrome patient, and does not synthesize p53 sinced a frame shiftmutation causes premature termination in the amino terminal region ofthe p53 molecule. MDAH041 were the cells of choice for thesetransfections because they do not express detectable levels of SDI1.They, therefore, provided a more sensitive assay for small changes inDNA synthesis inhibitory activity that might be expected in the case ofthese minimally deleted mutant constructs.

One μCi/ml tritiated thymidine was added to the culture medium 24 hoursafter transfection and the cells were incubated for an additional 36hours. The cells were fixed, stained for β-galactosidase activity, andprocessed for autoradiography to determine the percentage ofβ-galactosidase positive cells that had synthesized DNA. Percentinhibition was determined relative to control cells co-transfected withthe pCMV vector and pCMVB-gal.

Deletion of amino acids 53-58 was found to result in the greatest lossof DNA synthesis inhibitory activity (which was about 50% that of thefull length cDNA). Two other deletions (of amino acids 42-47 and ofamino acids 66-71 of SEQ ID NO:2) also caused a decrease in activity,although of lesser extent. Since the possibility existed that aparticular construct that was actually minimally inhibitory mightexhibit greater inhibition if a larger amount of DNA were transfected,the amount of plasmid DNA was reduced to 200 ng per transfection. Theresults of transfection experiments using this lower amount of DNAconfirmed that the deletion of amino acids 42-47 and 66-71 of SEQ IDNO:2 did indeed result in a loss of inhibitory activity, and that thedeletion of amino acids 53-58 of SEQ ID NO:2 resulted in a significantloss in ability to inhibit DNA synthesis. Deletions 24-29, 30-35 hadactivity similar to that of wild type at both DNA concentrations. Theseresults indicate that the critical region of the protein product of SDI1must lie between amino acids 42 and 71. They therefore support theconclusion that peptides having the sequence of SEQ ID NO:2₄₂₋₇₁ andnon-peptide molecules that mimic a reactive side group of SEQ IDNO:2₄₂₋₇₁ comprise mimetics of SDI-1. In particular, a preferred mimetichas the amino acid sequence SEQ ID NO:2₄₉₋₇₇. A particularly preferredpeptide mimetic has a sequence (SEQ ID NO:34):WNFDFXXXXPLEGXXXWXXVXXXXLPXXY. Such a mimetic may be formed using theabove-described methods and primers having the sequences:

SEQ ID NO:35 CAGAATCACAAGCCACTCGAGGGTAAG TACGAGTGGGAGCGTGTGCGGGGCCTT SEQID NO:36 CTTACCCTCGAGTGGCTTGTGATT CTGAAAGTCGAAGTTCCATCGCTC

A preferred nonpeptide mimetic has residues that mimic reactive sidegroups of SEQ ID NO:34.

To determine whether the DNA synthesis inhibition caused by thesemutants was occurring through inhibition of cdk kinases, the binding ofin vitro translated deletion mutant proteins products with purifiedcdk2protein was examined. Thus, the various deletion mutants of SDI-1were translated in reticulocyte lysates (Promega) using pBluescriptbased plasmids as transcription templates for the T7 RNA polymerase. Forin vitro binding, 45 μl of the translation product was added to 500 μlbinding buffer (50 mM Tris/HCl, pH 7.5, 120 mM NaC1, 2 mM EDTA, 0.1%NP-40, 1 mM NaF, 0.1 m.M sodium vanidate, 5 μg/ml leupeptin, 5 μg/mlsoybean trypsin inhibitor, 5 μg/ml aprotinin) containing 1 ug cdk2protein purified from a bacculovirus expression system. The mixture wasgently rocked for 1 hour at 40° C. and then 7.5 μg anti-cdk2 rabbitpolyclonal antibody was added. Mixing was continued for 1 hour at 4° C.The immunocomplex was absorbed by incubation with 40 μl protein G plus Abeads for 2 hr. The matrices were then washed three times with 0.5 ml ofbinding buffer prior to electrophoresis and autoradiography.

All the mutant constructs produced protein which had a molecular weightof approximately 23 kD. To verify that the proteins were indeed deletedSDI-1 products, they were immunoprecipitated using CA5 (a monoclonalantibody against the HA tag sequence) and all successfully precipitatedthe antibody. The wild type protein SDI-1 and the three deletions,24-29, 30-35, 72-77, bound cdk2 protein efficiently, suggesting that theDNA synthesis inhibitory activity of these constructs was the result ofbinding to cdk2. In contrast, the mutants, 42-47, 53-58, and 66-71,which had decreased inhibitory activity did not bind cdk2.

Immunohistochemistry

The intracellular localization of SDI-1 has been reported to be nuclearand there is a putative nuclear translocation signal at the 3′ end ofthe coding region of the gene. However, as indicated above, aC-terminus-truncated protein (SDI-1₁₋₇₁), which lacks this putativenuclear translocation signal was found to have the same inhibitoryactivity as the wild type protein (Table 13). In order to determine thelocalization of the protein products of the various deletion mutantconstructs of SDI-1 following their transfection into the MDAH 041cells, immunostaining of the HA tag was conducted.

For immunostaining, cells were seeded onto glass coverslips at about 50%confluent density, and allowed to adhere overnight before transfection.Following transfection cells were incubated at 37° C. for 24 hours, thenwashed twice with phosphate buffered saline (PBS) and fixed in freshlyprepared formaldehyde solution [4% (wt/vol) paraformaldehyde in PBS] for15 min at room temperature. The fixed cells were then washed in PBS andincubated in 50 mM glycine in PBS at room temperature for 10 min. Cellswere again washed with PBS and permeabilized by incubation in 0.2%Triton-X100 in PBS, at room temperature, for 10 min. After additionalwashings with PBS immunostaining was performed according to thedirection of the ABC kit (source). The primary antibody (12CA5) wasdiluted 1:1000 in the buffer available in the kit.

The deleted, but still growth inhibitory proteins were found to localizeto the nucleus whereas a high percentage of cells expressing theinactive or less active mutant proteins exhibited cytoplasmic staining.These data, along with the cdk binding results, strongly indicate thattranslocation of SDI-1 into the nucleus is dependent on the formation ofa SDI-1 cyclin-cdk complex.

Thus, truncation of the C terminal region of SDI-1 revealed that SDI-1molecules having amino acids 1-71 of SEQ ID NO:2 exhibited almost thesame capacity to inhibit DNA synthesis as the full length SDI-1 protein(SEQ ID NO:2). Fine deletion analysis showed that the amino acids inregion 42-71 of SEQ ID NO:2 were crucial for both kinase inhibition andDNA synthesis inhibition. This result was also confirmed by resultswhich showed that SDI-1 molecules having deletions of the amino acids inregion 42-71 of SEQ ID NO:2 exhibited no binding activity.

From immunohistochemical analysis, most of the active mutant protein wasfound to be localized in the nucleus. In contrast, SDI-1 mutants thatlacked activity were found to be localized in the cytoplasm, indicatingthat translocation of SDI-1 into the nucleus is mainly depend upon theability of making the complexes with cdk-cyclins rather than upon theexistence of nuclear translocation-like sequences on the C-terminus ofthe SDI-1 protein. Because of the significance of region 42-71 of SEQ IDNO:2 on kinase inhibition and DNA synthesis inhibition, this region wasstudied in great detail. This analysis clearly indicated that homologyregions (49-53 and 58-61 of SEQ ID NO:2) provided a new inhibitory motifamong cdk inhibitors (such as SDI-1 and p27).

The above results clearly implicate the amino terminal region of theSDI-1 protein as the area involved in inhibiting DNA synthesis. The finemapping studies implicate the region between amino acids 48-65 ascritical for the negative growth effects of the gene. The data alsodemonstrate that when the gene product is involved in active inhibitionit localizes to the nucleus of the cell and that inactive forms of theprotein remain in the cytoplasm. With respect to this latterobservation, it is of interest to note that a putative nuclearlocalization signal exists in the carboxy terminal region of theprotein. Nonetheless, the deletion of amino acids 72-164 whicheliminates this signal sequence, is fully capable of inhibiting DNAsynthesis and is expressed in the nucleus. This indicates that someother molecule(s), perhaps the complexes of cyclins, cdks and PCNA withwhich SDI-1 associates, tranports the SDI-1 protein the nucleus.

EXAMPLE 24 Liposome-Mediated Intracellular Delivery of SDI Molecules

The ability of liposomes to deliver SDI-1 molecules into recipient cellswas demonstrated using the B16 murine melanoma cell line. Purifiedprotein (25 μg) (either SDI-1 obtained as described above, or bovineserum albumin (BSA), used as a control) was incubated with 10 82 g ofcommercially obtained Lipofectamine™ liposomes (Life Technologies, Inc.)[formed from a 3:1 (w/w) mixture of the polycationic lipid DOSPA and theneutral lipid DOPE]. The protein was incubated in the presence of theLipofectamine™ for 15 minutes at room temperature. The incubationoccurred in a final volume of 0.2 ml serum-free medium. Such incubationis sufficient to permit the desired association of SDI-1 protein andliposome.

After the incubation period, the reaction mixture was added to 0.8 ml ofB19 cells, resuspended in serum-free medium. After 4-5 hours, theLipofectamine™/protein was removed and the cell incubation wascontinued. Cells were evaluated at various times for (1) the presenceand location of internalized SDI-1 (identified through the use oflabeled anti-SDI-1 antibody), (2) their capacity to mediate theincorporation of tritiated thymidine into their DNA, (3) cellularproliferation and (4) changes in cellular morphology. Cells for cellcounts and tritiated thymidine incorporation were trypsinized at 24hours post lipofection and replated at 2,500 cells per well of a 96-wellplate (for thymidine incorporation analysis) or 10,000 cells per well ofa 24-well plate for cell number determinations. The results of thisexperiment are shown in Table 15. Cells were evaluated for the presenceand location of the delivered protein. After 3-4 hours, SDI-1 could bedetected in the nuclei of ½ of the cells that had received SDI-1. After9 hours, SDI-1 could be detected in the nuclei of ⅓ of such cells. After48 hours, SDI-1 was no longer differentially observed in the nuclei ofsuch cells which exhibited a gray staining over the entire cell. SDI-1was not detectable 72 hours after liposome treatment. SDI-1 could not bedetected (at any time) in cells that had received the BSA control (orbuffer alone). After 48 hours, cells that had received SDI-1 appearedlarger than control cells; after 72 hours, such cells were much largerthan control cells. In Table 15, “Protein Delivered” indicates whetherthe liposomes were associated with the BSA control, or with the SDI-1protein; “SDI-1 Staining” indicates the observed presence, frequency andlocation of SDI-1 in the recipient cells; “Cell Number” provided thetiter of cells ± experimental error; “³H-Tdr Uptake” denotes the extentof incorporation of tritiated thymidine into the cell's DNA.

TABLE 15 Ratio of SDI-1 Treated to BSA Treated Protein Time Cell ³H-TdrCell ³H-Tdr Delivered (hr) Number Uptake No. Uptake SDI-1 48  7820 ± 141 2134 ± 294 0.29 0.34 BSA 48 26540 ± 2828  6187 ± 211 Buffer 48 16040 ±226  4057 ± 28 SDI-1 72 23710 ± 1485  8765 ± 533 0.28 0.3 BSA 72 8576028773 ± 810

The above experiment demonstrated the capacity of the liposomes tomediate the delivery of SDI-1 into the melanoma cells. The SDI-1 wasactive in such cells, since the number of cells and the capacity of thecells to take up tritiated thymidine was markedly reduced, in comparisonto BSA treated cells. The experiment further demonstrated the ability ofthe delivered SDI-1 protein to migrate to the nuclei of the melanomacells, and to persist for a therapeutically effective period of timewithin the nucleus. As indicated in Table 15, a significant change ingross cellular morphology occurred.

EXAMPLE 25 Enhanced Chemotherapeutic Ability of SDI Molecules inCombination with Conventional Antineoplastic Agents

The mechanism of action of an antineoplastic plant alkaloid, CPT, wasexplored in order to demonstrate the chemotherapeutic ability of SDImolecules, and in particular, the value of combining the administrationof a chemotherapeutic antineoplastic agent with that of an SDI molecule.

The plant alkaloid CPT and its derivatives have demonstrated highantiproliferative and toxic activity against a wide variety of humantumor cells in vitro and in vivo (Slichenmyer, W. J. et al., J. Natl.Canc. Inst. 85:271-291 (1993)). The mechanism of action of thesecompounds involves stabilization of the covalent adducts formed betweenDNA and the nuclear enzyme topoisomerase I, an event that leads tointerference with the breakage-reunion process of DNA strands (Liu, L.F., Ann. Rev. Biochem. 58:351-375 (1989); Potmesil, M. et al., Canc.Res. 54:1431-1439 (1994)). Other events associated with formation ofDNA-topo I-CPT complexes include inhibition of DNA replication,induction of expression of early-response genes, induction ofdifferentiation, and internucleosomal DNA fragmentation (Aller, P. etal., Canc. Res. 52:1245-1251 (1992)), a characteristic feature ofprogrammed cell death, or apoptosis (Umansky, S. R., In: Apoptosis: TheMolecular Basis of Cell Death, Tomei, L. D. et al. (eds), Cold SpringHarbor Laboratory, Cold Spring, N.Y., pp. 193-208 (1991); Wyllie, A. H.et al., In: Oxford Textbook of Pathology: Principles of Pathology,McGee, J.O.D. et al. (eds.), Oxford University Press, pp. 141-157(1992)).

The CPT derivative, 9NC, elicits perturbations in the cell cycle invitro that correlate with the ability of these cells to induce tumorswhen xenografted in nude mice (Pantazis, P. et al., Canc. Res.53:1577-1582 (1993); Pantazis, P. et al., Intl. J Canc. 53:863-871(1993); Pantazis, P. et al., Canc. Res. 54:771-776 (1994)).Specifically, 9NC-treated tumorigenic breast, ovarian and malignantmelanocytes die by apoptosis while traversing the S phase of the cellcycle (Pantazis, P. et al., Canc. Res. 53:1577-1582 (1993); Pantazis, P.et al., Intl. J Canc. 53:863-871 (1993); Pantazis, P. et al., Canc. Res.54:771-776 (1994)). Further, it appears that induction of apoptosis isirreversible in 9NC-treated tumorigenic cells, that is the 9NC-treatedcells die by apoptosis without requiring continuous presence of 9NC(Pantazis, P. et al., Canc. Res. 53:1577-1582 (1993); Pantazis, P. etal., Canc. Res. 54:771-776 (1994)). In comparison, non-tumorigenicbreast and ovarian epithelial cells treated with 9NC accumulate at theS-G₂ boundary of the cell cycle and only a small number of cells dyingby apoptosis. The extent of cell accumulation in S-G₂ boundarycorrelates with the length of 9NC treatment and/or 9NC concentration(Pantazis, P. et al., Canc. Res. 53:1577-1582 (1993); Pantazis, P. etal., Intl. J Canc. 53:863-871 (1993); Pantazis, P. et al., Canc. Res.54:771-776 (1994)). The sensitivity of malignant cells to CPT and itsderivatives has been correlated positively with topo I synthesis andactivity, and/or drug-induced accumulation of DNA-topo I-CPT complexes(Liu, L. F., Ann. Rev. Biochem. 58:351-375 (1989); Potmesil, M. et al.,Canc. Res. 54:1431-1439 (1994)).

Recent reports suggest that CPT may alter p34^(cdc2)/cyclin B complexregulation in HeLa cells (Tsao, Y. P. et al., Canc. Res. 52:1823-1829(1992)) and induce wild type p53 protein in ML-1 myeloid leukemia cellsand in LNCaP prostatic adenocarcinoma cells (Nelson, W. G. et al.,Molec. Cell. Biol. 14:1815-1823 (1994)). Significantly, both eventsappear to require active DNA synthesis. In this context, it has beenreported that p53 may activate wild type p53-activated fragment 1, SDI-1(sometimes referred to as Waf-1) El-Deiry, W. S. et al., Cell 75:805-816(1993)). As indicated above, SDI-1 plays a critical role in theregulation of cell growth in tumor and senescent cells by inhibitingcyclin-dependent kinases and by subsequently interrupting the celldivision process. To expand these observations and to gain insight intothe molecular mechanism of CPT-induced cytostasis, the expression ofSDI-1 was evaluated in non-tumorigenic cells and the results werecorrelated with those from studies examining the effects of CPT on cellproliferation and metabolic activity, DNA synthesis, and perturbation inthe cell cycle.

A. Characteristic Effects of 9NC

The effect of 9NC (5 to 80 nM) on cell proliferation and DNA synthesiswas evaluated by the Cell Proliferation Kit-XTT (Boehringer MannheimCorp., IN), a non-radioactive, colorimetric ELISA assay which reflectsmitochondrial dehydrogenase activity. The assay is based on the cleavageof the yellow tetrazolium salt XTT to form an orange formazan dye bydehydrogenase activity in active mitochondria. This conversion occursonly in living cells and correlates with their overall metabolicactivity.

Human hepatoblastoma cells, HepG2 cells were obtained from the AmericanType Culture Collection and maintained in RPMI-1640 medium (GIBCO, GrandIsland, N.Y.) supplemented with 10% fetal bovine serum (GIBCO), 100units/ml of penicillin, 1009 μg/ml of streptomycin, 250 mg/ml ofamphotericin (Sigma Chemical Co.), and 4 mM L-glutamine (GIBCO). Thecell cultures were incubated at 37° C. in a humidified CO₂ (5%)atmosphere. The cell number in untreated and 9NC-treated cultures wasdetermined with the aid of cell counter (Coulter Electric, Inc.,Hialeah, Fla.) and cell viability was monitored by trypan blue dyeexclusion.

9NC was semisynthesized from CPT and purified as described by Wani, M.C. et al. (J. Med. Chem. 29:2358-2363 (1986)). A fine suspension of 9NC(10 μM) was prepared in polyethylene glycol (PEG 400; Aldrich,Milwaukee, Wis.), divided into small aliquots and stored at −80° C.until used. 9NC was always added 24 h after cell plating.

DNA synthesis was measured by 5-bromo-2′-deoxy-uridine (BrdU)incorporation into cellular DNA following 24 h and 72 h treatment with9NC using the Cell Proliferation Assay Kit-BrdU/ELISA (BoehringerMannheim Corp.). Inhibition of DNA synthesis correlated with the lengthof 9NC treatment and the concentration of 9NC in the cell culture.Importantly, whereas only moderate suppression of metabolic activity(approximately 20% was observed in 72 h cultures treated with 20 nM of9NC, at least four-fold decrease in DNA synthesis was demonstrated incorresponding cultures. Furthermore, in accordance with previousobservation that active DNA synthesis is required for 9NC-induced cellarrest, a more prominent inhibitory effect of 9NC was observed in cellcultures treated with 9NC for 72 h as compared to cultures treated for24 h.

The inhibition of DNA synthesis correlated with at least a two-folddecrease in cell numbers following 72 h treatment at 20 nM concentrationof 9NC. This inhibitory effect was not associated with cell death asassessed by trypan blue dye exclusion. Taken together, these resultsindicate that low doses of 9NC (20 nM) induce significant inhibition ofcell proliferation and DNA synthesis without affecting metabolicactivity of the cells.

Phase contrast microscopy of 9NC-treated cells revealed an increasedcell size (2 to 5-fold) with prominent cytoplasmic compartment. After 3to 5 days of treatment the morphology of the cells remained stable withvery little change during the three to four week culture period.Overall, cells appeared to be healthy and metabolically active, andresembled the morphology of primary human hepatocytes grown on plasticsurfaces. Virtually no cell proliferation was observed following removalof 9NC and the cells maintained a differentiated phenotype withoutgrowth.

In addition, attempts to subculture the cells failed. Such an absence ofsubstantial growth is a characteristic feature of senescent and/orhighly differentiated cells.

The proportions of cells in the different phases of the cell cycle wereinvestigated by monitoring changes in the relative DNA content of thecells using flow cytometry following the methodology of Pantazis, P. etal. (Canc. Res. 53:1577-1582 (1993); Intl. J Canc. 53:863-871 (1993)).The changes in the DNA content were monitored both as a function oftreatment period and drug concentration and compared with the resultsobtained in untreated cultures. Untreated cell cultures contain arelative high percentage of cell fraction in G₂, and a small butdetectable fraction of hyperdiploid cells. Overall, little changeoccurred in DNA content at different incubation periods and equaldistributions of the cells in G₁ and G₂ phases of the cycle wereobserved. A 24 h treatment of the cells with increasing concentrationsof 9NC resulted in a decrease in G₁-fraction, and a concomitant increasein the S+G₂ and hyperdiploid fraction. The histogram area on the leftside of the G₂-peak indicates that more cells are arrested in and/ortraverse the S phase as the 9NC concentration increases. Higher 9NCconcentrations resulted in hyperdiploid cells that had a DNA contentwhich was less than that of hyperdiploid cells generated by the presenceof lower 9NC concentrations.

Like 24 h-treated cultures, cultures treated for 72 h and 120 hcontained hyperdiploid cell fractions with decreasing DNA content as the9NC-concentration increases. Further, a relative increase in theapoptotic fraction was observed that correlates with increases induration of drug treatment and concentration. Removal of 9NC fromcultures treated for 48 h or more did not significantly alter theresults described above indicating that the effects of 9NC on the cellswere irreversible. It should be noted that the findings described inthis section were observed in cells attached on the plastic substratethat were trypsin-removed for studies.

B. The Antineoplastic Characteristics of 9NC are Attributable to itsInduction of SDI-1

The preceding experiments determined that 9NC at low doses (up to 20 nM)induced cytostatic effect without associated cell death by apoptosis ornecrosis. Therefore, initial studies to evaluate the levels of SDI-1were performed with low doses of 9NC. Total RNA was isolated from HepG2cells treated with 5 nM and 20 nM 9NC for 72 h and the levels of SDI-1mRNAs expression in cells were examined by Northern blot hybridizationanalysis. Whereas no or little change was observed in the expression ofGAPDH, Northern analysis demonstrated a dose dependent increase in SDI-1expression in 9NC-treated cells. The relative amount of SDI-1 mRNA ineach sample was calculated as the ratio of SDI-1/GAPDH. At least 3-foldincrease in SDI-1 expression was observed when 20 nM of 9NC was used.

Caffeine abolishes G₂ cell cycle checkpoint induced by DNA-damagingagents (Traganos, F. et al., Canc. Res. 53:4613-4618 (1993); Lau, C. C.et al., Proc. Natl. Acad. Sci. (U.S.A.) 79:2942-2946 (1982)). To assessthe specificity of SDI-1 induction by 9NC, the levels of SDI-1expressions were evaluated via Northern blot analysis after simultaneousaddition of caffeine (1 mM) and 9NC. Whereas simultaneous addition ofcaffeine and 9NC abrogated the 9NC-dependent induction of SDI-1expression, caffeine alone, or addition of caffeine after 24 hincubation with 9NC had no appreciable effect (of 9NC) on SDI-1expression. Further, no differences in the levels of SDI-1 expressionwere observed between 24 h and 72 h treatment with 9NC, suggesting thatinduction of SDI-1 is an early event. Likewise, no changes in the levelsof SDI-1 were observed following removal of 9NC. These resultscorrelated with data obtained using flow cytometry by monitoring changesin the relative DNA content of the cells treated in the same conditions.To establish the kinetics of 9NC-induced upregulation of SDI-1 gene, thecells cultured for 24 h in control medium were treated with 20 nM of 9NCfor 0.5 h, 1.5 h, 3 h, 6 h, 12 h, and 24 h respectively. Gene expressionincreased gradually and reached maximum at 24 h. Little changes wereobserved in corresponding 0 h, 3 h, 6 h, and 24 h control cell cultures,although, gradual decreases in the levels of SDI-1 expression throughthe duration of the experiment were noted.

Treatment of non-tumorigenic hepatoblastoma cells with 9NC thus resultedin a dose-dependent inhibition of cell proliferation and DNA synthesis.This effect correlated with at least a two-fold decrease in cell numbersin 9NC-treated cultures when compared to cell numbers in untreatedcultures and was not associated with cell death as assessed by trypanblue dye exclusion when low doses of 9NC were used. Significantly, onlya moderate decrease in overall metabolic activity (approximately 20%)was observed when 20 nM of 9NC was used despite at least 2-fold decreasein cell numbers and at least 4-fold decrease in DNA synthesis. Thisresult indicates that 20 nM of 9NC induces a cytostatic state withoutaffecting the metabolic activity of the cells. Light and electronmicroscopy further confirmed these findings and revealed enlarged cellswith prominent cytoplasmic compartments containing generous lysosomes,mitochondria and accumulation of lipid in the cytoplasm.

The data thus indicate that low doses of 9NC induce a cytostatic ratherthan cytotoxic effect. Furthermore, both morphological and growthcharacteristics such as decreased capability to divide even afterremoval of 9NC resembles growth characteristics of cultured primaryhepatocytes and the phenomenon of cellular senescence. These results areconsistent with recently published data that described CPT-induceddifferentiation and stimulation of the expression ofdifferentiation-related genes in U-937 cells (Aller, P. et al., Canc.Res. 52:1245-1251 (1992)). Flow cytometry revealed that the majority ofthe cells were arrested in the G₂ phase of the cell cycle. Takentogether, these results indicate that 9NC at low concentrations causescell arrest in a hepatoblastoma cells in G₂ phase and inducesmorphologic and growth features of highly differentiated or senescentcells.

In sum, non-tumorigenic immortal hepatoblastoma cells, HepG2, arrestedat the G₂ phase of the cell cycle by 9NC, exhibited morphologic,ultrastructural and growth characteristics of senescent cells andoverexpressed SDI-1 mRNA. Induction of SDI-1 by 9NC demonstrates thatSDI-1 can be overexpressed in the G₂ phase of the cell cycle andconfirms the conclusion that genes upregulated in senescent cells arealso induced in arrested cancer cells. The results further demonstratedthat at least a three-fold increase in SDI-1 gene expression was inducedby 20 nM 9NC in hepatoblastoma cells. Since a consequence of 9NCadministration is the production of SDI-1, the co-administration ofSDI-1 and 9NC would permit a therapeutic effect to be obtained usinglower doses of 9NC. Thus, the results of the above example support theconclusion that the use of SDI molecules in combination with “standard”antineoplastic agents can increase the effectiveness of anticancertherapy.

EXAMPLE 26 Enhanced Chemotherapeutic Ability of SDI Molecules inCombination with Conventional Antineoplastic Agents

SDI-1 appears to prevent the inactivation by phosphorylation of the rbgene product (pRb). One aspect of the present invention concerns therecognition that SDI-1 inhibits the activity of E2F, agrowth-stimulatory transcription factor that is negatively regulated byunphosphorylated pRb.

pRb is underphosphorylated in quiescent cells, where it is thought tosuppress growth. As cells approach S phase, pRb is progressivelyphosphorylated, at least in part by cdks, which inactivate its growthsuppressive properties (Buchkovich, K. et al., Cell 58:1097-1105 (1989);Chen, P. L. et al., Cell 58:1193-1198 (1989); Mihara, K. et al., Science246:1300-1303 (1989); DeCaprio, J. A. et al., Proc. Natl. Acad. Sci.(U.S.A.) 89:1795-1798 (1992)). Underphosphorylated pRb binds a varietyof proteins. One of these is E2F (Chellappan, S. P. et al., Cell65:1053-1061 (1991); Helin, K. et al., Cell 70:337-350 (1992); Kaelin,W. G. et al., Cell 70:351-364 (1992); Shan, B. et al., Molec. Cell.Biol. 12:5620-5631 (1992)), a transcription factor whose activity isrequired for the expression of several genes at the G1/S boundary(Farnham, P. J. et al., Biochim. Biophys. Acta 1155:125-131 (1993)). pRbphosphorylation in the late G1 is believed to release E2F, which isneeded for activity). Thus, entry into S phase appears to be regulatedby the sequential activation of cdks which lead to a progressivephosphorylation of pRb and release of E2F, which, in turn, stimulatesthe transcription of genes needed for DNA replication and mediates entryinto S phase.

The effect of the human SDI-1 on transcription driven by twoE2F-responsive promoters was determined. Proliferating A31 cells weretransfected with an SDI-1 expression vector in which the SDI-1 cDNA wasdriven by the strong constitutive cytomegalovirus early promoter (CMV)together with a reporter vector in which the chloramphenicolacetyltransferase (CAT) gene was driven by either the hamsterdihydrofolate reductase (DHFR) promoter or the human cdc2 promoter(cdc2-CAT). A31 cells are Balb/c murine 3T3 fibroblasts whose origin andgrowth properties have been described (Lu, K. et al., Molec. Cell. Biol.9:3411-3417 (1989); Dimri, G. P et al., J. Biol. Chem. 269:16180-16186(1994), herein incorporated by reference). Cells were transfected with 2μg of expression vectors, reporters vectors (for transactivation) andthe CMV-β-gal identification/normalization vector using Lipofectamine(Gibco-BRL), as described by Dimri, G. P et al. (J. Biol. Chem.269:16180-16186 (1994), herein incorporated by reference). The vectorbackbone was added to the mixture as needed to equalize the amount oftransfected DNA. Seventy-two hours later, the cells were lysed andassayed for chloramphenicol acetyl transferase (CAT) and β-galactosidaseactivity. Both DHFR and cdc2 were expressed by proliferating cells,largely due to E2F-mediated transcription that is dependent on E2Fbinding sites in the 5′ regulatory regions of these genes (Slansky, J.E. et al., Molec. Cell. Biol. 13:1610-1618 (1993); Dalton, S., EMBO J.35 11:1797-1804 (1992)).

The SDI-1 expression vector, CMV-SDI-1, but not the control vector,suppressed CAT activity driven by either the DHFR or cdc2 promoter(Table 16). CMV-SDI-1 was maximally inhibitory at above 2 μg of DNAunder the assay conditions used. CAT activity in Table 16 is reported incounts per minute, normalized to the β-galactosidase activity. At thisconcentration, CMV-SDI-1 inhibited the DHFR promoter, which contains twostrong E2F binding sites (Slansky, J. E. et al., Molec. Cell. Biol.13:1610-1618 (1993)), somewhat more strongly than the cdc2 promoter,which contains a single E2F binding site (Dalton, S., EMBO J.11:1797-1804 (1992)) (50-70% inhibition vs 40-50% inhibition). Thisdifference in sensitivity is consistent with the finding that the DHFRpromoter is somewhat more sensitive than the cdc2 promoter tostimulation by E2F1, a component of E2F (Dimri, G. P et al., J. Biol.Chem. 269:16180-16186 (1994)). At the concentrations used here,CMV-SDI-1 inhibited DHFR-CAT and cdc2-CAT expression to approximatelythe same extent that it inhibited DNA replication.

TABLE 16 Expression Normalized Report Vector CAT % % Vector cDNAActivity Activity Inhibition DHFR-CAT None* 9462 100 0 SDI-1 9155 97 40.2 μg 1.0 8196 87 13 2.0 2613 28 72 4.0 1757 19 81 cdc2-CAT None* 3304100 0 SDI-1 1664 50 50 1.0 μg 2.0 2000 60 40 4.0 2008 61 39 SV-CAT None*3620 100 0 SDI-1 3743 103 0 1.0 μg 2.0 3828 106 0 4.0 3366 93 7 fos-CATNone* 16927 100 0 SDI-1 16273 100 0 2.0 μg

In sharp contrast to the response of the DHFR and cdc2 promoters,CMV-SDI-1 had little or no effect on reporter activity driven by eitherthe SV 40 early promoter (SV-CAT or the c-fos promoter (fos-CAT) (Table16). Neither the SV40 early genes nor c-fos has been reported to beE2F-responsive.

To more critically ascertain whether the inhibition of transcription bySDI-1 was E2F-dependent, three E2F binding sites were inserted into theSV40 early promoter and the effect of CMV-SDI-1 on CAT activity drivenby this modified promoter (SV/E2F) was determined. To constructSV/E2F-CAT and SV/E2Fm-CAT, three copies of the sequence encompassingthe E2F binding site in the dihydrofolate reductase promoter wasinserted into the Bgl II site that is just upstream of the SV40 earlypromoter in SV-CAT. The E2F binding site was wild-type in SV/E2F-CAT,and carried a CGCGCC to CGATCC mutation within the 12 residue longbinding site (Slansky, J. E. et al., Molec. Cell.

Biol. 13:1610-1618 (1993); Nevins, J. R., Science 258:424-429 (1992)).

CMV-SDI-1 (2 μg) suppressed the activity of SV/E2F-CAT to about the sameextent that it inhibited the activity of DHFR-CAT. By contrast, mutantE2F binding sites, similarly inserted into the SV40 promoter(SV/E2Fm-CAT), did not confer SDI-1-suppressible CAT activity. ThusSDI-1 can inhibit transcription that is dependent on a wild-type, butnot mutant, E2F site.

Cotransfection of an E2F1 expression vector (CMV-E2F1) with CMV-SDI-1almost completely reversed the inhibition of DNA synthesis.SDI-1-mediated suppression of DNA synthesis was also reversed by an SV40large T antigen expression vector. SV 40 T antigen binds and inactivatesboth the p53 and pRb tumor suppressor proteins, as well as thepRb-related proteins p107 and p130 (Fanning, E. et al., Ann. Rev.Biochem. 61:55-85 (1992)). A mutant T antigen carrying a small deletionthat inactivates the p53-binding function (T[p]) was about 70% aseffective as wild-type T antigen in reversing growth inhibition bySDI-1. The T[p] expression vector was constructed by inserting into CMV1the BamHI-Bgl II fragment of Td1434-444, which contains the SV40 earlyregion bearing the deletion corresponding to amino acids 434-444 inlarge T antigen.

By contrast, a mutant T antigen carrying a point mutation thatinactivated the pRb and pRb-related protein binding function (T[K])failed to reverse SDI-1-mediated suppression of DNA synthesis. Inaddition to reversing SDI-1's antiproliferative effects, CMV-E2F1 alsoreversed SDI-1-dependent inhibition of E2F transaction, as judged by itseffect on DHFR-CAT and cdc2-CAT expression. Taken together, the resultsindicate that SDI-1 is capable of inhibiting E2F transactivationactivity, and that this inhibition appears to be important for SDI-1'santiproliferative effects.

To further demonstrate the conclusion that the ability of SDI-1 toinhibit growth was closely linked to its ability to inhibitE2F-dependent transcription, the inhibitory properties of several SDI-1mutants were examined. These mutants included a naturally occurringSDI-1 variant (p21 pm) in which the serine at amino acid 31 was replacedby an arginine. This polymorphism has been detected in approximately 10%of normal human cells thus far examined, and appears to result in aslight reduction in the antiproliferative properties of SDI-1 (Table17). A SDI-1 expression vector for this allele (CMV-p21 pm) was comparedwith CMV-SDI-1 for ability to inhibit DNA synthesis and E2F-mediatedtransactivation in proliferating A31 cells. Both SDI-1 and the SDI-1polymorphic protein suppressed growth and transcription driven by thecdc2 and SV/E2F promoters, but riot the SV/E2Fm promoter. In all cases,however, CMV-SDI-1 pm was 5-10% less effective than CMV-SDI-1 atinhibiting both DNA synthesis and E2F-mediated transaction (Table 17).

Several SDI-1 deletion mutants were also tested for their ability toinhibit DNA synthesis and DHFR-driven CAT activity. Expression vectorsencoding SDI-1 proteins carrying deletions in amino acids 24 to 29, 53to 58, or 17 to 52 were introduced into proliferating A31 cells, andtheir ability to inhibit DNA synthesis and DHFR-driven CAT activity werecompared. There was a good correspondence between the extents ofdeletion and the mutants' relative abilities to inhibit DNA synthesisand DHFR-driven transcription. In Table 17, “% LN” denotes thepercentage of transfected (β-galactosidase positive) cells withradiolabeled nuclei; “SDI-1” denotes an expression vector that expressesthe wild-type SDI-1 protein, “p21 pm” denotes an expression vector thatexpresses the above-discussed SDI-1 polymorphic mutant protein.

TABLE 17 Transcription DNA Normalized Synthesis Reporter Expression % %% Vector Vector cDNA Activity Inhibition % LN Inhibition SV/E2F- None100 0 68 0 CAT SDI-1 50 50 17 75 p21pm 53 47 25 63 SV/E2Fm- None 100 0CAT SDI-1 93 7 p21pm 82 18 cdc2-CAT None 100 0 SDI-1 52 48 p21pm 62 38

Because SDI-1 inhibits the protein kinases that phosphorylate pRb, andunphosphorylated pRb is believed to negatively regulate E2F activity(Nevins, J. R., Science 258:424-429 (1992)), pRb is presumed to be acentral mediator of SDI-1's effects. Since A31 cells express a normal Rbgene, the ability of SDI-1 to inhibit E2F activity, and possibly growth,in tumor cells that lack a functional pRb was tested. For this purpose,the ability of SDI-1 to inhibit DNA synthesis and E2F activity in C33A(HTB 31), a human cervical carcinoma cell line that does not express afunctional pRb by virtue of a frameshift mutation was evaluated. C33Acells were obtained from the American Type Culture Collection(Rockville, Md.).

C33A cells were found to be clearly sensitive to both the growthinhibitory and transcription inhibitory properties of SDI-1. Indeed,SDI-1 had a substantial antiproliferative effect on C33A cells, althoughthey were somewhat less sensitive than A31 cells (40-50% inhibition vs65-75% inhibition). By contrast, C33A and A31 cells were equallysensitive to SDI-1 inhibition of E2F activity. Thus, CMV-SDI-1 reducedDHFR-, cdc2- and SV/E2F-driven CAT activity to approximately equalextents in C33A and A31 cells. As expected CMV-SDI-1 had no effect onSV/E2Fm-driven CAT activity in either cell line. Taken together, theresults demonstrate that SDI-1 can act to inhibit DNA synthesis and E2Factivity through cellular targets other than pRb.

E2F binding sites are occupied by protein complexes, whosecomposition—at least by in vitro analyses—depend on the cellular growthstate and stage of the cell cycle (Chellappan, S. P. et al., Cell65:1053-1061 (1991); Nevins, J. R., Science 258:424-429 (1992); Farnham,P. J. et al., Biochim. Biophys. Acta 1155:125-131 (1993)). To determinewhich if any of these complexes might be a target for SDI-1, the effectof recombinant SDI-1 protein on DNA-protein complexes that associatewith an E2F binding site in vitro was determined.

Nuclear extracts from quiescent cells contain several E2F complexes,despite the fact that E2F1, the cell cycle regulated component of E2Fthat is a target for pRb binding (Helin, K. et al., Cell 70:337-350(1992); Kaelin, W. G. et al., Cell 70:351-364 (1992); Shan, B. et al.,Molec. Cell. Biol. 12:5620-5631 (1992)), is not expressed (Shan, B. etal., Molec. Cell. Biol. 12:5620-5631 (1992); Slansky, J. E. et al.,Molec. Cell. Biol. 13:1610-1618 (1993); Dimri, G. P et al., J. Biol.Chem. 269:16180-16186 (1994)). Quiescent cell extracts contain only lowlevels of “free” E2F, the presumed active form of E2F, but also verylittle E2F in association with pRb. Rather, quiescent cells contain E2Fcomponents in association with the pRb-related protein p107, and/or inassociation with p130 (Cobrinik et al., Genes Devel 7:2392-2404 (1993)).None of the E2F complexes present in quiescent cell extracts were foundto be affected the addition of SDI-1 protein. In addition, none of theE2F complexes in quiescent cells were disrupted or supershifted by aSDI-1 antibody.

E2F-1 is induced a few hours prior to S phase (Shan, B. et al., Molec.Cell. Biol. 12:5620-5631 (1992); Slansky, J. E. et al., Molec. Cell.Biol. 13:1610-1618 (1993); Dimri, G. P et al., J. Biol. Chem.269:16180-16186 (1994)) at about which time nuclear extracts from cellsin mid to late G1 contain increased levels of free E2F, E2F inassociation with pRb, and E2F in association with p107, cdk2 and cyclinE (mid-late G1-early S). Of the complexes present in extracts from cellsin late G1, only the E2F/p107/cdk/cyclin complexes were disrupted byrecombinant SDI-1.

In quiescent cells, SDI-1 expression is high; E2F activity is low, withlow levels of free E2F and E2F/p107/cdk/cyc complex. Mitogens suppressSDI-1 expression, leading to induction of E2F1 mRNA; this in turninduces free E2F, E2F/p107/cdk/cycE, and ultimately DNA synthesis. Thefinding that SDI-1 does not affect complex formation in G0 may reflecteither that, in G0, E2F/p107 is not associated with cyclin/cdk.Alternatively, the finding may indicate that all E2F activity in G0 isassociated with p130, and that SDI-1 has no effect on p130/E2F with orwithout cyclins.

Antisense SDI-1 artificially lowers SDI-1 expression in Q cells, andthis leads to induction of E2F1 expression, free E2F, E2F/p107/cdk/cyccomplex, and DNA synthesis. Growing cells express high levels of SDI-1,E2F1 expression, E2F/p107/cdk/cyc complex, and E2F activity. Inductionof SDI-1, as might occur in responded to damage or elevated p53,disrupts E2F/p107/cdk/cyc complexes, which may mediate rapid growtharrest. If SDI-1 levels remain high, E2F1 expression declines, followedby a decline in free E2F and establishment of quiescent state.

As indicated by the above experiments, SDI-1 was found to inhibitE2F-mediated transcription activation. Moreover, SDI-1 mutants thatfailed to inhibit E2F also failed to inhibit DNA replication, and E2F-1,the cell cycle regulated component of E2F, antagonized the inhibition oftransaction and DNA replication by SDI-1. However, pRb was not essentialfor these effects: SDI-1 inhibited growth and E2F activity inpRb-deficient cells. SDI-1 selectively disrupted a DNA binding complexcontaining E2F, cdk2, cyclin E or A and the Rb-related protein p107 invitro (and in cells, and in cells, SDI-1 downregulated the level of E2F1mRNA). We conclude that SDI-1 inhibits growth at least in part bysuppressing E2F activity, possibly by disrupting the E2F/cdk2/p107complex to elicit a rapid growth arrest and, ultimately, causing adeficiency of E2F-1. The results also indicate pRb is not essential forgrowth suppression by SDI-1, and suggest that in Rb-deficient cellsSDI-1 may act through pRb-related proteins such as p107 or p130.

The recognition of molecular mechanisms through which SDI-1 mediates itsbiological activity permits the use of directed screening assays toidentify mimetics and antagonists of SDI-1 and its binding partners.Such assays can be used to produce SDI molecules having more desiredcharacteristics.

EXAMPLE 27 In vivo Ability to Deliver SDI-1 to Skin Cells: Ability ofSDI-1 to Inhibit Hair Growth

As indicated above, SDI-1 is capable of inhibiting cellularproliferation and of establishing a quiescent state when delivered toactively proliferating recipient cells. Conversely, the administrationof an inhibitor of SDI-1 permits a quiescent cell to regain itsproliferative capacity.

The ability of SDI-1 to prevent hair loss was determined bydemonstrating the capacity of SDI-1 to be delivered to hair folliclesand to mediate a change in the capacity of the hair follicles to formhair.

Hair growth was assayed using the Dermatek™ histoculture in vitrohistoculture assay system (AntiCancer, San Diego, Calif.). The Dermatek™histoculture assay system is described by Li, L. et al. (In Vitro CellDevel Biol. 28A:479-481 (1992); In Vitro Cell Devel Biol. 28A:695-98(1992), both references herein incorporated by reference).

The Dermatek™ system exploits the fact that the activity of hairfollicles is governed by a continuous cycle composed of a telogen (nohair growth) phase, an anagen (hair growth phase) phase and a catagenphase, and a return to the telogen phase. Mice that are in the telogenphase are induced to enter the anagen phase by depilation. Since, inmice, all truncal melanocytes are confined to the hair follicles, theproduction of melanin is strictly coupled to the anagen phase of thefollicular cycle. Thus, depilation permits one to synchronize thefollicle cycle. By employing mice such as C57BI6, the progression of thehair follicle into the anagen phase can be followed by monitoring theproduction of melanin. Thus, the skin of such mice change fromwhite/pink (telogen), to gray (mid-anagen), to black (late anagen). Inthe Dermatek™ assay, biopsies of skin are suspended oncollagen-containing sponge-gel supports and cultured under in vivo-likeconditions. The assay thus provides large numbers of homogeneous, maturefollicles of defined hair stage.

For such assays, SDI-1, prepared as described above, was incorporated(via sonication) into liposomes having a composition ofphosphatidylcholine, cholesterol, and phosphatidylethanolamine (ratio of5:3:2). The ratio of lipid to SDI-1 was 10:1.

C57BI6 mice were depilated with wax/rosin as described by Li, L. et al.(In Vitro Cell Devel Biol. 28A:479-481 (1992); In Vitro Cell Devel Biol.28A:695-698 (1992)). Four days after depilation, 4 pieces of 4 mmacu-punched skin tissues were removed and placed into organ culture.Twenty-four hours later, a liposome preparation containing either 100μg/ml, 50 μg/ml, or 30 μg/ml of SDI-1 was topically administered to theskin cultures. The ability of SDI-1 to inhibit melanin production andhair growth in skin areas that had received the liposome preparation wasdetermined by photo-microscopy 24 and 48 hours after SDI-1 liposomeadministration.

Significant hair follicle development and melanin production was evidentin the skin of the depilated animals 5 days after depilation. Melaninproduction was intense. Control animals evaluated 24 hours laterexhibited even more substantial melanin production and hair folliclegrowth. Both the number and size of melanin-producing foci hadincreased. Melanin production appeared as intense foci adjacent toregions of less intense, more diffuse areas of pigmentation.

Twenty-four hours after being treated with the liposome preparationcontaining 30 μg/ml of SDI-1, the skin of C57BI6 mice exhibited asignificant reduction in both the number of developing hair folliclesand the extent of melanin production. Melanin production was not asintense as in the control animals, and some foci exhibited only minimalpigmentation.

C57BI6 mouse skin treated with the liposome preparation containing 50μg/ml of SDI-1 exhibited (24 hours after treatment) an even moresubstantial decrease in follicle growth and melanin production than thatexhibited by the mouse skin treated with 30 μg/ml SDI-1 liposomes. Onlya small number of foci appeared to have any intensity of melaninproduction, and the exhibited production of such foci was markedly lessthan those of control or 30 μg/ml SDI treated animals. The majority offoci exhibited little or no follicle development, and minimal anddiffuse melanin production.

A still greater decrease in follicle growth and melanin production wasobserved in the skin of C57BI6 mice treated with 100 μg/ml SDI-1liposomes at 24 hours post treatment.

The responses were also found to persist over time. Thus, whereascontrol mice at day 7 (i.e. 48 hours after the experimental mice hadreceived SDI-1 liposomes) exhibited pronounced intense melaninproduction and hair follicle development, mice that had received SDI-1liposomes exhibited continued inhibition of melanin production andfollicle growth.

Mouse skin that had received the liposome preparation containing 30μg/ml of SDI-1, skin after 48 hours exhibited increased melaninproduction compared to 24 hours, thus indicating the transiency of thehair growth inhibition over time. Although the extent of melaninproduction after 48 hours for such mouse skin exceeded that observedafter 24 hours, the amount of melanin produced was substantially lessthan that observed in the untreated controls.

Such transiency was not observed in mouse skin that had been treatedwith liposome preparations containing either 50 μg/ml of SDI-1 or 100μg/ml of SDI-1, thus indicating that the levels of SDI-1 in the hairfollicles of such animals had not yet decayed below a therapeutic level,or that the administration had a prolonged effect.

The experiment demonstrates several aspects of the present invention:

(1) It demonstrates the capacity to deliver SDI-1 to skin follicles viaa liposome formulation.

(2) It demonstrates that the delivered SDI-1 was biologically active,and hence that the it was not merely delivered to cells, in vivo, butwas moreover capable of migrating intracellularly to its appropriatetarget site.

(3) The experiment demonstrates the ability of SDI-1 to cause theactively proliferating cells of hair follicles to cease (in a doseresponsive manner) producing products (hair and melanin) that areassociated with such proliferation. Thus, the experiment demonstrates anin vivo ability of SDI-1 to induce quiescence in proliferating cells.

(4) The experiment indicates that SDI-1 would be an effective agent inthe treatment of hair loss induced by chemotherapy. As indicated above,alopecia (hair loss) is an undesired side effect of chemotherapy. Thehair loss reflects the damage or death of hair follicles due to thepresence of the chemotherapeutic agent. The eventual restoration of hairproduction in patients who have undergone chemotherapy evidences therepair or replacement of such follicles. Since the above-describedexperiment demonstrates the ability of SDI-1 to induce the cells of hairfollicles into a quiescent state, the administration of SDI-1 to thehair follicles of patients who are to undergo chemotherapy would beexpected to minimize or prevent hair loss due to such chemotherapy. Suchadministration would be expected to transiently prevent new hair growth(as indicated in the experiment) while simultaneously inhibiting DNAsynthesis. The inhibition of DNA synthesis permits DNA repair processesto repair or rescue cells of the hair follicles that have been damagedby the chemotherapeutic agent. Upon recovery from the SDI-1administration, the cells of the hair follicles (which are also nolonger being exposed to the chemotherapeutic agent) reinitiate thesynthesis of hair. Thus, overall, the patient experiences a transientcessation of new hair growth, but does not experience the hair losspreviously associated with such chemotherapy.

(5) The experiment demonstrates that the administration of SDI-1provides a reversible transient therapy whose therapeutic effect may besustained by either increasing the dose of administered SDI-1, or byproviding additional doses.

(6) The experiment demonstrates the capacity of SDI-1 to retard and/ortransiently prevent hair growth, and thus indicates the ability of SDI-1to be used to prevent undesired (facial, leg, etc.) hair growth or totreat hirsutism.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this Application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

36 2106 base pairs nucleic acid single linear cDNA NO NO Homo sapiensSENESCENT HUMAN CELLS SENESCENT CELL DERIVED CDNA LIBRARY SDI-1 1CCTGCCGAAG TCAGTTCCTT GTGGAGCCGG AGCTGGGCGC GGATTCGCCG AGGCACCGAG 60GCACTCAGAG GAGGCGCCAT GTCAGAACCG GCTGGGGATG TCCGTCAGAA CCCATGCGGC 120AGCAAGGCCT GCCGCCGCCT CTTCGGCCCA GTGGACAGCG AGCAGCTGAG CCGCGACTGT 180GATGCGCTAA TGGCGGGCTG CATCCAGGAG GCCCGTGAGC GATGGAACTT CGACTTTGTC 240ACCGAGACAC CACTGGAGGG TGACTTCGCC TGGGAGCGTG TGCGGGGCCT TGGCCTGCCC 300AAGCTCTACC TTCCCACGGG GCCCCGGCGA GGCCGGGATG AGTTGGGAGG AGGCAGGCGG 360CCTGGCACCT CACCTGCTCT GCTGCAGGGG ACAGCAGAGG AAGACCATGT GGACCTGTCA 420CTGTCTTGTA CCCTTGTGCC TCGCTCAGGG GAGCAGGCTG AAGGGTCCCC AGGTGGACCT 480GGAGACTCTC AGGGTCGAAA ACGGCGGCAG ACCAGCATGA CAGATTTCTA CCACTCCAAA 540CGCCGGCTGA TCTTCTCCAA GAGGAAGCCC TAATCCGCCC ACAGGAAGCC TGCAGTCCTG 600GAAGCGCGAG GGCCTCAAAG GCCCGCTCTA CATCTTCTGC CTTAGTCTCA GTTTGTGTGT 660CTTAATTATT ATTTGTGTTT TAATTTAAAC ACCTCCTCAT GTACATACCC TGGCCGCCCC 720CTGCCCCCCA GCCTCTGGCA TTAGAATTAT TTAAACAAAA ACTAGGCGGT TGAATGAGAG 780GTTCCTAAGA GTGCTGGGCA TTTTTATTTT ATGAAATACT ATTTAAAGCC TCCTCATCCC 840GTGTTCTCCT TTTCCTCTCT CCCGGAGGTT GGGTGGGCCG GCTTCATGCC AGCTACTTCC 900TCCTCCCCAC TTGTCCGCTG GGTGGTACCC TCTGGAGGGG TGTGGCTCCT TCCCATCGCT 960GTCACAGGCG GTTATGAAAT TCACCCCCTT TCCTGGACAC TCAGACCTGA ATTCTTTTTC 1020ATTTGAGAAG TAAACAGATG GCACTTTGAA GGGGCCTCAC CGAGTGGGGG CATCATCAAA 1080AACTTTGGAG TCCCCTCACC TCCTCTAAGG TTGGGCAGGG TGACCCTGAA GTGAGCACAG 1140CCTAGGGCTG AGCTGGGGAC CTGGTACCCT CCTGGCTCTT GATACCCCCC TCTGTCTTGT 1200GAAGGCAGGG GGAAGGTGGG GTCCTGGAGC AGACCACCCC GCCTGCCCTC ATGGCCCCTC 1260TGACCTGCAC TGGGGAGCCC GTCTCAGTGT TGAGCCTTTT CCCTCTTTGG CTCCCCTGTA 1320CCTTTTGAGG AGCCCCAGCT ACCCTTCTTC TCCAGCTGGG CTCTGCAATT CCCCTCTGCT 1380GCTGTCCCTC CCCCTTGTCC TTTCCCTTCA GTACCCTCTC AGCTCCAGGT GGCTCTGAGG 1440TGCCTGTCCC ACCCCCACCC CCAGCTCAAT GGACTGGAAG GGGAAGGGAC ACACAAGAAG 1500AAGGGCACCC TAGTTCTACC TCAGGCAGCT CAAGCAGCGA CCGCCCCCTC CTCTAGCTGT 1560GGGGGTGAGG GTCCCATGTG GTGGCACAGG CCCCCTTGAG TGGGGTTATC TCTGTGTTAG 1620GGGTATATGA TGGGGGAGTA GATCTTTCTA GGAGGGAGAC ACTGGCCCCT CAAATCGTCC 1680AGCGACCTTC CTCATCCACC CCATCCCTCC CCAGTTCATT GCACTTTGAT TAGCAGCGGA 1740ACAAGGAGTC AGACATTTTA AGATGGTGGC AGTAGAGGCT ATGGACAGGG CATGCCACGT 1800GGGCTCATAT GGGGCTGGGA GTAGTTGTCT TTCCTGGCAC TAACGTTGAG CCCCTGGAGG 1860CACTGAAGTG CTTAGTGTAC TTGGAGTATT GGGGTCTGAC CCCAAACACC TTCCAGCTCC 1920TGTAACATAC TGGCCTGGAC TGTTTTCTCT CGGCTCCCCA TGTGTCCTGG TTCCCGTTTC 1980TCCACCTAGA CTGTAAACCT CTCGAGGGCA GGGACCACAC CCTGTACTGT TCTGTGTCTT 2040TCACAGCTCC TCCCACAATG CTGATATACA GCAGGTGCTC AATAAACGAT TCTTAGTGAA 2100AAAAAA 2106 164 amino acids amino acid linear protein NO NO HOMO SAPIENSSDI-1 Senescent cell derived cDNA library 2 Met Ser Glu Pro Ala Gly AspVal Arg Gln Asn Pro Cys Gly Ser Lys 1 5 10 15 Ala Cys Arg Arg Leu PheGly Pro Val Asp Ser Glu Gln Leu Ser Arg 20 25 30 Asp Cys Asp Ala Leu MetAla Gly Cys Ile Gln Glu Ala Arg Glu Arg 35 40 45 Trp Asn Phe Asp Phe ValThr Glu Thr Pro Leu Glu Gly Asp Phe Ala 50 55 60 Trp Glu Arg Val Arg GlyLeu Gly Leu Pro Lys Leu Tyr Leu Pro Thr 65 70 75 80 Gly Pro Arg Arg GlyArg Asp Glu Leu Gly Gly Gly Arg Arg Pro Gly 85 90 95 Thr Ser Pro Ala LeuLeu Gln Gly Thr Ala Glu Glu Asp His Val Asp 100 105 110 Leu Ser Leu SerCys Thr Leu Val Pro Arg Ser Gly Glu Gln Ala Glu 115 120 125 Gly Ser ProGly Gly Pro Gly Asp Ser Gln Gly Arg Lys Arg Arg Gln 130 135 140 Thr SerMet Thr Asp Phe Tyr His Ser Lys Arg Arg Leu Ile Phe Ser 145 150 155 160Lys Arg Lys Pro 19 base pairs nucleic acid single linear cDNA NO YESHOMO SAPIENS 3 AGCCGGTTCT GACATGGCG 19 12 amino acids amino acid linearpeptide NO N-terminal [His]6 leader peptide 4 Met Arg Gly Ser His HisHis His His His Gly Ala 1 5 10 699 base pairs nucleic acid single linearcDNA NO NO Schistosoma japonicum GST 5 ATGTCCCCTA TACTAGGTTA TTGGAAAATTAAGGGCCTTG TGCAACCCAC TCGACTTCTT 60 TTGGAATATC TTGAAGAAAA ATATGAAGAGCATTTGTATG AGCGCGATGA AGGTGATAAA 120 TGGCGAAACA AAAAGTTTGA ATTGGGTTTGGAGTTTCCCA ATCTTCCTTA TTATATTGAT 180 GGTGATGTTA AATTAACACA GTCTATGGCCATCATACGTT ATATAGCTGA CAAGCACAAC 240 ATGTTGGGTG GTTGTCCAAA AGAGCGTGCAGAGATTTCAA TGCTTGAAGG AGCGGTTTTG 300 GATATTAGAT ACGGTGTTTC GAGAATTGCATATAGTAAAG ACTTTGAAAC TCTCAAAGTT 360 GATTTTCTTA GCAAGCTACC TGAAATGCTGAAAATGTTCG AAGATCGTTT ATGTCATAAA 420 ACATATTTAA ATGGTGATCA TGTAACCCATCCTGACTTCA TGTTGTATGA CGCTCTTGAT 480 GTTGTTTTAT ACATGGACCC AATGTGCCTGGATGCGTTCC CAAAATTAGT TTGTTTTAAA 540 AAACGTATTG AAGCTATCCC ACAAATTGATAAGTACTTGA AATCCAGCAA GTATATAGCA 600 TGGCCTTTGC AGGGCTGGCA AGCCACGTTTGGTGGTGGCG ACCATCCTCC AAAATCGGAT 660 CTGGTTCCGC GTGGATCCCC GGGAATTCATCGTGACTGA 699 232 amino acids amino acid linear protein NO Schistosomajaponicum GST 6 Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu ValGln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr GluGlu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys LysPhe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp GlyAsp Val Lys 50 55 60 Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala AspLys His Asn 65 70 75 80 Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu IleSer Met Leu Glu 85 90 95 Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser ArgIle Ala Tyr Ser 100 105 110 Lys Asp Phe Glu Thr Leu Lys Val Asp Phe LeuSer Lys Leu Pro Glu 115 120 125 Met Leu Lys Met Phe Glu Asp Arg Leu CysHis Lys Thr Tyr Leu Asn 130 135 140 Gly Asp His Val Thr His Pro Asp PheMet Leu Tyr Asp Ala Leu Asp 145 150 155 160 Val Val Leu Tyr Met Asp ProMet Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175 Val Cys Phe Lys Lys ArgIle Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185 190 Leu Lys Ser Ser LysTyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195 200 205 Thr Phe Gly GlyGly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg 210 215 220 Gly Ser ProGly Ile His Arg Asp 225 230 13 base pairs nucleic acid single linearcDNA NO NO linker fragment for GST-SDI-1 gene fusion 7 GGATCCCCCC GCC 1310 base pairs nucleic acid single linear cDNA NO NO linker fragment forGST-SDI-1 gene fusion 8 CCCTCGAGGG 10 7 amino acids amino acid linearpeptide NO internal hinge region of GST-SDI-1 fusion protein 9 Pro ArgGly Asp Pro Pro Ala 1 5 1194 base pairs nucleic acid single linear cDNANO NO GST-SDI-1 gene fusion 10 ATGTCCCCTA TACTAGGTTA TTGGAAAATTAAGGGCCTTG TGCAACCCAC TCGACTTCTT 60 TTGGAATATC TTGAAGAAAA ATATGAAGAGCATTTGTATG AGCGCGATGA AGGTGATAAA 120 TGGCGAAACA AAAAGTTTGA ATTGGGTTTGGAGTTTCCCA ATCTTCCTTA TTATATTGAT 180 GGTGATGTTA AATTAACACA GTCTATGGCCATCATACGTT ATATAGCTGA CAAGCACAAC 240 ATGTTGGGTG GTTGTCCAAA AGAGCGTGCAGAGATTTCAA TGCTTGAAGG AGCGGTTTTG 300 GATATTAGAT ACGGTGTTTC GAGAATTGCATATAGTAAAG ACTTTGAAAC TCTCAAAGTT 360 GATTTTCTTA GCAAGCTACC TGAAATGCTGAAAATGTTCG AAGATCGTTT ATGTCATAAA 420 ACATATTTAA ATGGTGATCA TGTAACCCATCCTGACTTCA TGTTGTATGA CGCTCTTGAT 480 GTTGTTTTAT ACATGGACCC AATGTGCCTGGATGCGTTCC CAAAATTAGT TTGTTTTAAA 540 AAACGTATTG AAGCTATCCC ACAAATTGATAAGTACTTGA AATCCAGCAA GTATATAGCA 600 TGGCCTTTGC AGGGCTGGCA AGCCACGTTTGGTGGTGGCG ACCATCCTCC AAAATCGGAT 660 CTGGTTCCGC GTGGATCCCC TCGAGGGGATCCCCCCGCCA TGTCAGAACC GGCTGGGGAT 720 GTCCGTCAGA ACCCATGCGG CAGCAAGGCCTGCCGCCGCC TCTTCGGCCC AGTGGACAGC 780 GAGCAGCTGA GCCGCGACTG TGATGCGCTAATGGCGGGCT GCATCCAGGA GGCCCGTGAG 840 CGATGGAACT TCGACTTTGT CACCGAGACACCACTGGAGG GTGACTTCGC CTGGGAGCGT 900 GTGCGGGGCC TTGGCCTGCC CAAGCTCTACCTTCCCACGG GGCCCCGGCG AGGCCGGGAT 960 GAGTTGGGAG GAGGCAGGCG GCCTGGCACCTCACCTGCTC TGCTGCAGGG GACAGCAGAG 1020 GAAGACCATG TGGACCTGTC ACTGTCTTGTACCCTTGTGC CTCGCTCAGG GGAGCAGGCT 1080 GAAGGGTCCC CAGGTGGACC TGGAGACTCTCAGGGTCGAA AACGGCGGCA GACCAGCATG 1140 ACAGATTTCT ACCACTCCAA ACGCCGGCTGATCTTCTCCA AGAGGAAGCC CTAA 1194 397 amino acids amino acid linearprotein NO GST-SDI-1 fusion protein 11 Met Ser Pro Ile Leu Gly Tyr TrpLys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu TyrLeu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly AspLys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn LeuPro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln Ser Met Ala IleIle Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys ProLys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala Val Leu Asp IleArg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110 Lys Asp Phe Glu ThrLeu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Leu Lys MetPhe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp HisVal Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 ValVal Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg210 215 220 Gly Ser Pro Arg Gly Asp Pro Pro Ala Met Ser Glu Pro Ala GlyAsp 225 230 235 240 Val Arg Gln Asn Pro Cys Gly Ser Lys Ala Cys Arg ArgLeu Phe Gly 245 250 255 Pro Val Asp Ser Glu Gln Leu Ser Arg Asp Cys AspAla Leu Met Ala 260 265 270 Gly Cys Ile Gln Glu Ala Arg Glu Arg Trp AsnPhe Asp Phe Val Thr 275 280 285 Glu Thr Pro Leu Glu Gly Asp Phe Ala TrpGlu Arg Val Arg Gly Leu 290 295 300 Gly Leu Pro Lys Leu Tyr Leu Pro ThrGly Pro Arg Arg Gly Arg Asp 305 310 315 320 Glu Leu Gly Gly Gly Arg ArgPro Gly Thr Ser Pro Ala Leu Leu Gln 325 330 335 Gly Thr Ala Glu Glu AspHis Val Asp Leu Ser Leu Ser Cys Thr Leu 340 345 350 Val Pro Arg Ser GlyGlu Gln Ala Glu Gly Ser Pro Gly Gly Pro Gly 355 360 365 Asp Ser Gln GlyArg Lys Arg Arg Gln Thr Ser Met Thr Asp Phe Tyr 370 375 380 His Ser LysArg Arg Leu Ile Phe Ser Lys Arg Lys Pro 385 390 395 24 base pairsnucleic acid single linear cDNA NO NO Primer 12614 12 GGAGGATCCATGTCAGAACC GGCT 24 24 base pairs nucleic acid single linear cDNA NO NOPrimer 12615 13 GCAGAATTCC TGTGGGCGGA TTAG 24 20 base pairs nucleic acidsingle linear cDNA NO NO Primer 14 TCTAGGCCTG TACGGAAGTG 20 60 basepairs nucleic acid single linear cDNA NO NO Primer 15 TAGGAATTCACTAGTCTAAG CGTAATCTGG AACATCGTAT GGGTAGGGCT TCCTCTTGGA 60 30 base pairsnucleic acid single linear cDNA NO NO Primer 16 TTCGGCCCTC GAGGCCTGAGCCGCGACTGT 30 30 base pairs nucleic acid single linear cDNA NO NO Primer17 GCTCAGGCCT CGAGGGCCGA AGAAGCGGCG 30 31 base pairs nucleic acid singlelinear cDNA NO NO Primer 18 TTAGCGCGCC TCGAGGCTGC TCGCTGTCCA C 31 31base pairs nucleic acid single linear cDNA NO NO Primer 19 CGAGCAGCCTCGAGGCGCGC TAATGGCGGG C 31 30 base pairs nucleic acid single linear cDNANO NO Primer 20 GGCTGCCCTC GAGGCCGATG GAACTTCGAC 30 30 base pairsnucleic acid single linear cDNA NO NO Primer 21 CCATCGGCCT CGAGGGCAGCCCGCCATTAG 30 36 base pairs nucleic acid single linear cDNA NO NO Primer22 CGTGAGCGAC CCCGGGGCGT CACCGAGACA CCACTG 36 36 base pairs nucleic acidsingle linear cDNA NO NO Primer 23 CTCGGTGACG CCCCGGGGTC GCTCACGGGCCTCCTG 36 30 base pairs nucleic acid single linear cDNA NO NO Primer 24TTCGACCCTC GAGGCCTGGA GGGTGACTTC 30 30 base pairs nucleic acid singlelinear cDNA NO NO Primer 25 CTCCAGGCCT CGAGGGTCGA AGTTCCATCG 30 36 basepairs nucleic acid single linear cDNA NO NO Primer 26 ACCGAGACATCCCGGGCCGA CTTCGCCTGG GAGCGT 36 36 base pairs nucleic acid single linearcDNA NO NO Primer 27 GGCGAAGTCG GCCCGGGATG TCTCGGTGAC AAAGTC 36 36 basepairs nucleic acid single linear cDNA NO NO Primer 28 CCACTGGAGCCCCGGGGCCG TGTGCGGGGC CTTGGC 36 36 base pairs nucleic acid single linearcDNA NO NO Primer 29 CCGCACACGG CCCCGGGGCT CCAGTGGTGT CTCGGT 36 30 basepairs nucleic acid single linear cDNA NO NO Primer 30 GCCTGGCCTCGAGGCGGCCT GCCCAAGCTC 30 30 base pairs nucleic acid single linear cDNANO NO Primer 31 CAGGCCGCCT CGAGGCCAGG CGAAGTCACC 30 41 base pairsnucleic acid single linear cDNA NO NO Primer 32 CGGGGCCTTC CCCGGGGCCTTCCCACGGGG CCCCGGCGAG G 41 40 base pairs nucleic acid single linear cDNANO NO Primer 33 CGTGGGAAGG CCCCGGGGAA GGCCCCGCAC ACGCTCCCAG 40 29 aminoacids amino acid linear peptide NO internal peptide mimetic fragment 34Trp Asn Phe Asp Phe Xaa Xaa Xaa Xaa Pro Leu Glu Gly Xaa Xaa Xaa 1 5 1015 Trp Xaa Xaa Val Xaa Xaa Xaa Xaa Leu Pro Xaa Xaa Tyr 20 25 54 basepairs nucleic acid single linear cDNA NO NO Primer 35 CAGAATCACAAGCCACTCGA GGGTAAGTAC GAGTGGGAGC GTGTGCGGGG CCTT 54 48 base pairsnucleic acid single linear cDNA NO NO Primer 36 CTTACCCTCG AGTGGCTTGTGATTCTGAAA GTCGAAGTTC CATCGCTC 48

What is claimed is:
 1. A liposome preparation that comprises an SDI-1protein consisting of the amino acid sequence SEQ ID NO:2.
 2. Theliposome preparation of claim 1 that comprises: (a) a mixture of apolycationic and a neutral lipid; and (b) and SDI-1 protein consistingof the amino acid sequence of SEQ ID NO:2.
 3. The liposome preparationof claim 2, wherein said polycationic lipid is2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium-trifluoroacetate(DOSPA).
 4. The liposome preparation of claim 2, wherein said neutrallipid is dioleolyphosphatidylethanolamine (DOPE).
 5. The liposomepreparation of claim 3, wherein said neutral lipid isdioleolyphosphatidylethanolamine (DOPE).
 6. The liposome preparation ofclaim 5, wherein said polycationic and a neutral lipid are present insaid liposome in a 3:1 (w/w) mixture.
 7. A method for preparing aliposome preparation of SDI molecules which comprises incubatingliposomes that comprise a mixture of a polycationic and a neutral lipidwith an SDI-1 protein consisting of the amino acid sequence of SEQ IDNO:2.